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The effect of etched 3D printed Cu-bearing titanium alloy on the polarization of macrophage

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3D printed titanium alloys have been widely used as implants in orthopedic surgery and dentistry. In recent years, Cu-bearing titanium alloys have shown great advantages in tissue engineering due to their excellent antibacterial activity and biological effect. In the current study, three alloys, namely, TC4 alloy, TC4-5Cu alloy, and TC4-6Cu alloy were fabricated by the use of selective laser melting (SLM) technology. Acid etching treatment was used to remove the metal powders on the samples and modify the surface of the manufactured alloys. The effect of different etched alloys on the biological behavior of macrophages (RAW 264.7) was studied comprehensively. Results showed that acid etching had no effect on the hydrophilicity, while contributing to the adhesion and polarization of macrophages with a lower ROS level. Moreover, Cu-bearing titanium exhibited better cell adhesion, macrophage polarization potential, and a lower ROS level. In summary, acid etching treatment provided a promising strategy to improve the biological properties of the Cu-bearing titanium alloys by SLM.
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The effect of etched 3D printed
Cu-bearing titanium alloy on the
polarization of macrophage
Jinge Yan
1
, Wanyi Huang
1
, Hai Kuang
2
*, Qiang Wang
1
and
Bo Li
1
*
1
School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of
Oral Disease, Shenyang, China,
2
Department of Oral and Maxillofacial Surgery, College of
Stomatology, Guangxi Medical University, Nanning, China
3D printed titanium alloys have been widely used as implants in orthopedic
surgery and dentistry. In recent years, Cu-bearing titanium alloys have shown
great advantages in tissue engineering due to their excellent antibacterial
activity and biological effect. In the current study, three alloys, namely,
TC4 alloy, TC4-5Cu alloy, and TC4-6Cu alloy were fabricated by the use of
selective laser melting (SLM) technology. Acid etching treatment was used to
remove the metal powders on the samples and modify the surface of the
manufactured alloys. The effect of different etched alloys on the biological
behavior of macrophages (RAW 264.7) was studied comprehensively. Results
showed that acid etching had no effect on the hydrophilicity, while contributing
to the adhesion and polarization of macrophages with a lower ROS level.
Moreover, Cu-bearing titanium exhibited better cell adhesion, macrophage
polarization potential, and a lower ROS level. In summary, acid etching
treatment provided a promising strategy to improve the biological properties
of the Cu-bearing titanium alloys by SLM.
KEYWORDS
3D printing, copper, Ti6Al4V, acid etching, macrophage, ROS, oxidative stress
1 Introduction
As a common disease of oral surgery, maxillofacial bone defect caused by tumors,
trauma, and other reasons affects the patientsappearance and directly harms the
swallowing, chewing, and conversing functions of patients. In recent years, there have
been two main approaches to repairing maxillofacial bone defects: surgical reconstruction
and bone tissue engineering. However, surgical reconstruction has relative limitations in
clinical application. Hence, bone tissue engineering plays an important role in
maxillofacial bone repair. As one of the general bone tissue engineering technologies,
3D printing technology has been widely used in clinical practice in recent years due to its
excellent characteristics, such as the ability to achieve personalized precise designs, be
produced quickly and efciently, and reduce the time and risk of surgical exposure
(Dawood et al., 2015).
OPEN ACCESS
EDITED BY
Hasan Uludag,
University of Alberta,
REVIEWED BY
Ying Zhao,
Shenzhen Institutes of Advanced
Technology, (CAS),
Fatemeh Kabirian,
Faculty of Medicine, KU Leuven,
*CORRESPONDENCE
Hai Kuang,
kuanghai@hotmail.com
Bo Li,
bli24@cmu.edu.cn
SPECIALTY SECTION
This article was submitted to
Biomaterials,
a section of the journal
Frontiers in Materials
RECEIVED 11 May 2022
ACCEPTED 27 June 2022
PUBLISHED 10 August 2022
CITATION
Yan J, Huang W, Kuang H, Wang Q and
Li B (2022), The effect of etched 3D
printed Cu-bearing titanium alloy on the
polarization of macrophage.
Front. Mater. 9:941311.
doi: 10.3389/fmats.2022.941311
COPYRIGHT
© 2022 Yan, Huang, Kuang, Wang and
Li. This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms.
Frontiers in Materials frontiersin.org01
TYPE Original Research
PUBLISHED 10 August 2022
DOI 10.3389/fmats.2022.941311
3D printing, also called additive manufacturing (AM), is a
quick advanced technology that creates solid models by adding
powder layer by layer. This technology has already been used for
maxillofacial bone defects, dental defects, and dentition defects
(Javaid and Haleem, 2019). The commonly used biomaterials are
metals, alloys, ceramics, and polymers. These materials are used
in different medical disciplines according to the requirements of
specic applications. Previous research illustrated that metal
materials were the most commonly used materials in dentistry
due to their excellent mechanical properties (strength, hardness,
wear resistance, durability, toughness, etc.) compared with
ceramic materials and resin materials (Palaskar et al., 2010).
Clinicians in the eld of dentistry and orthopedic surgery
demand materials with good mechanical properties, corrosion
resistance, and biocompatibility, and titanium alloys largely meet
these requirements.
In recent years, different metal elements are added to
titanium alloys to improve the properties of the alloys. As one
of the necessary micro-elements for the body, copper is a cofactor
of various enzymes involved in the life activities of the body. In
addition, copper is also widely used in various biomaterial
research studies due to its excellent antimicrobial activity and
biological activity (Zhang et al., 2016). Carter et al. (Carter et al.,
2017) have veried that copper-bearing implants can inhibit the
activity of both Gram-positive and Gram-negative bacteria.
Huang et al. (Huang et al., 2019) demonstrated that copper-
containing titanium alloy has good biocompatibility, and copper
ions released from its surfaces could act as an inammatory
regulator to promote osteogenesis and sterilization.
A retrospective study on failed dental implants by Sun et al.
(Sun et al., 2017) showed that 47% of early implant failures were
caused by inammation. Peri-implant diseases and infections
have become the focus of oral implantology prevention and
treatment (Spriano et al., 2018). Research studies have proven
that compared with stainless steel materials (Chen et al., 2015)
and poly-ether-ether-ketone (PEEK) (Olivares-Navarrete et al.,
2015), titanium and titanium alloys have a lower level of
inammation and less abundance of macrophages on the
surfaces. The implant-related inammatory response can be
summarized in the following eight steps: exudation, protein
surface adsorption, growth of a temporary provisional matrix
based on blood, the capacity of cells of the innate immune system
to proliferate (white blood cells, platelets, complement, and blood
coagulation system), neutrophil migration, by replacing the
differentiation of the mononuclear cell into macrophages,
foreign body reaction, generation of reactive oxygen species,
and monocyte/macrophage fusion to form foreign body giant
cells or apoptosis. Among them, reactive oxygen species
production is an important step in the process of
inammatory response.
Reactive oxygen species (ROS) are cooperative or
independent regulators of cellular signaling in response to
different environmental stimuli and are mainly derived from
superoxide anions (O
2
), hydrogen peroxide (H
2
O
2
), and
hydroxyl radicals (OH
)(Mittal et al., 2014). It has been
reported that ROS are involved in DNA repair, cell cycling,
cell differentiation, chromatin remodeling, self-renewal, and
other cell processes by Rendra et al. (Rendra et al., 2019).
Furthermore, ROS plays an essential role in the regulation of
macrophage polarization. A reduced ROS level suppresses the
M1 phenotype and promotes macrophage polarization into the
M2 phenotype (Zhou et al., 2018). However, the link between
ROS and alloy-induced macrophage polarization has not been
well-claried.
Macrophages play a crucial role in the process of
inammation. Macrophages are able to secrete cytokines,
chemokines, and growth factors to attract broblasts to
produce extracellular matrix (ECM) and collagen. In addition,
macrophages express different functional procedures by
polarization according to different micro-environmental
signals (McWhorter et al., 2015). Macrophages have two
specic phenotypes, either M1-type macrophages (typically
pro-inammatory macrophages activated in response to
Th1 cellderived cytokines) or M2-type macrophages (anti-
inammatory macrophages activated in response to Th2 cell-
derived cytokines) (Zhang et al., 2018).
In the process of 3D medical printing of titanium alloy, there
may be partially melted or un-melted powder left on the surface
of the material, which may lead to residual particle pollution in
the later stage. Yi et al. (Yi et al., 2022) showed that acid etching
treatment of porous Ti alloy scaffolds could remove residual
powder on the surface of scaffolds and signicantly improve the
osteogenesis properties.
This study combines etched and unetched 3D printed Cu-
bearing titanium with RAW 264.7 cells to explore the effect of
macrophage biological behaviors, including the occurrence of an
alloy being in direct contact with cells and alloy extracting culture
macrophages. It could provide the experimental basis for animal
and clinical use of this surface treatment technology.
2 Materials and methods
2.1 Material preparation
Plate samples with a size of 10 × 10 × 2 mm were fabricated
by 3D printing technology. Selective laser melting (SLM) is one of
the rapidly developed additive manufacturing techniques that
creates solid models by adding powder layer by layer. The
fabrication detail can be found in the previous studies (Zong
et al., 2020); (Liu et al., 2021). According to a previous study, a
Class 1 laser system equipped with a ProX DMP 200 ber laser (a
wavelength of 1070 nm and maximum output power of 300 W)
was used to prepare Ti6Al4V-5Cu and Ti6Al4V-6Cu alloys
(Zong et al., 2020). Laser power, scanning speed, scanning
spacing, and single layer thickness are the major parameters
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in the process of selective laser melting. Different laser processing
parameters were compared, and the optimal parameters were
selected in this study: laser power of 260 W, scanning distance of
45 µm, and laser spot diameter of 70 µm. Then, all the samples
were ground with waterproof SiC paper from 800 to 2000 grits.
The specimens were subsequently immersed in nitric
acidhydrogen uoride acid solution and nally washed with
deionized water, acetone, and anhydrous ethanol for 10 min and
dried by cold air. The ratio between nitric acid, uoride acid, and
distilled water is 1:4:5, and the etching duration is 30 s. The
experimental samples were divided into six groups: unetched
TC4 (TC4), unetched TC4-5Cu (TC4-5Cu), unetched TC4-6Cu
(TC4-6Cu), etched TC4 (ETC4), etched TC4-5Cu (ETC4-5Cu),
and etched TC4-6Cu (ETC4-6Cu). The preparation of the extract
is according to ISO 1099312 standards. Figure 1 illustrates the
ow diagram of the study.
2.2 Surface characterization
2.2.1 Morphology
The structural morphology of the experimental samples was
tested by using a scanning electron microscope (SEM, Zeiss
Merlin Compact, Germany) equipped with energy-dispersive
spectroscopy (EDS).
2.2.2 Hydrophilicity measurement
The surface wettability of the alloys was detected by the
wettability measuring instrument (DataPhysics Instruments
Gmbh, Germany) with the droplet method. The initial volume
of the droplet is about 2 µl. SCA20 software was used to calculate
the contact angles on the left and right sides of the samples. Three
different areas were measured on each sample surface, and the
process was repeated once.
2.3 Behaviors of RAW 264.7 cells on
different material surfaces
2.3.1 Cell culture
Murine macrophage RAW 264.7 cells were used in this study. The
cells were cultured in Dulbeccosmodied Eaglesmedium(DMEM,
Hyclone, United States) supplemented with 10% FBS (Gibco) in an
atmosphere of 5% CO
2
at 37°C. The cells were passaged at
approximately 80% conuence and used at early passages (P5). The
preparation of extracts included immersing the plate samples of etched
and unetched titanium alloys in a cell culture medium for 72 h at a ratio
of 0.2 g/ml according to ISO 1099312 standard. After that, the extracts
were collected, ltered by a 0.2-µm lter, and stored at 4°C.
2.3.2 Cell proliferation and cytotoxicity assay
RAW 264.7 cell proliferation was analyzed by the Cell
Counting Kit-8 (CCK-8) for 12 and 24 h. RAW 264.7 cells
were cultured at a concentration of 2×10
4
cells/ml and then
seeded on all samples in a 96-well culture plate (500 µl/well, 37°C,
5% CO
2
). The extract was changed the next day. After incubation
for 12 and 24 h, respectively, the culture media was removed and
100 µl of DMEM medium with 10% CCK-8 (Beyotime, China)
was added. Then, the samples were incubated in a dark incubator
for 2 h at 37°C. Afterward, the 450 nm optical density (OD) was
obtained with an Enzyme standard instrument (Multiskan GO,
Thermo Scientic, United States ). The cell relative growth rate
(RGR) was calculated according to the following formula:
RGR ODexperimental groupODcontrol group ×100% .
2.3.3 Cell adhesion
RAW264.7 cells were seeded on different material surfaces at
a concentration of 2×10
4
cell/ml for 24 h. After incubation, the
medium was removed and the cells on alloys were washed three
FIGURE 1
Flow diagram of the study: (A) Specimen preparation; (B) Physical performance; (C) Behaviours of RAW264.7 cells on different alloy surfaces.
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times with phosphate-buffered saline (PBS) and xed with 4%
(w/v) paraformaldehyde for 10 min. And then, they were rinsed
twice by PBS again and permeabilized with 0.2% (v/v) Triton X-
100 (Beyotime, China) for 6 min. After that, 50 µL of rhodamine-
phalloidin (Molecular, Probes Thermo Fisher Scientic, China)
was added to stain cells in darkness. Finally, 5 mg/ml 40.6-
diamidino-2-phenylindole (DAPI) solution was added to stain
cells in darkness. The photographed stain images were observed
by an optical microscope.
2.3.4 Cell morphology
RAW 264.7 cells were seeded in confocal dishes at a
concentration of 2 × 10
4
cells/ml and cultured in different
kinds of material extracts for 24 h. After incubation, the
medium was removed and the cells were washed three
times with phosphate-buffered saline (PBS) and xed with
4% (w/v) paraformaldehyde for 10 min. Then, they were
rinsed twice by PBS again and permeabilized with 0.2% (v/
v) Triton X-100 (Beyotime, China) for 6 min. After that, 50 µl
rhodaminephalloidin (Molecular, Probes Thermo Fisher
Scientic, China) was added to stain cells in darkness.
Finally, 5 mg/ml Hoechst 33,342 (Boster, China) solution
was added to stain cells in darkness. The photographed
stain images were observed by a laser scanning confocal
microscope.
2.3.5 Flow cytometry test
A total of 1 × 10
6
RAW 264.7 cells were collected and
washed with PBS three times after being cultured in different
material extracts for 12 and 24 h. APC-conjugated
CD86 antibody (0.312 μg/test, Thermo Fisher Scientic) and
PE-conjugated CD206 antibody (0.625 μg/test, Thermo Fisher
Scientic) were incubated with the macrophages for 30 min on
ice. Finally, PBS was added to the tubes to keep the nal volume
at 200300 μLforow cytometry (BD Pharmingen, San
Diego, CA).
2.3.6 Measurement of intracellular reactive
oxygen species
The intracellular ROS production was determined by a
reactive oxygen species assay kit (Beyotime Biotechnology
Ltd., Shanghai, China) for 12 and 24 h. RAW 264.7 cells were
collected and incubated with DCFH-DA (1:1,000) for 30 min.
The cells were washed twice with PBS, and uorescence intensity
was monitored by a ow cytometer (Becton Dickinson,
America).
2.3.7 Statistical analysis
All experiments were analyzed with SPSS 23.0 software. The
results are presented as mean ± standard deviation. Differences
between groups were analyzed using one-way analysis of variance
(ANOVA) followed by Tukeys test. pvalues less than 0.05 were
considered statistically signicant.
3 Result
3.1 Material preparation
A class 1 laser system equipped with a ProX DMP 200 ber
laser (a wavelength of 1070 nm and maximum output power of
300 W) was used to prepare Ti6Al4V-5Cu and Ti6Al4V-6Cu
alloys, and the optimal parameters were selected in this study:
laser power of 260W, scanning distance of 45 µm, and laser spot
diameter of 70 µm (Zong et al., 2020).
According to ISO 1099312 guidelines, the preparation of extracts
included immersing the plate samples of etched and unetched titanium
alloys in a cell culture medium for 72 h at a ratio of 0.2 g/ml. An
analytical balance is used to measure the weight of each sample
(Table 1). The average weight of the TC4 alloy, TC4-5Cu alloy, and
TC4-6Cu alloy before etching and after etching are 0.0200g, 0.0236 g,
and 0.0281 g, respectively, and there is no statistical difference (p>0.05).
3.2 Characterizations of alloy
3.2.1 Surface morphology
Figure 2 shows the microstructures of the TC4 alloy surface,
TC4-5Cu alloy surface, and TC4-6Cu alloy surface before and after
acid etching technology. From the macro-graph of material samples
(Figure 2A), the surface of each alloy sample after grinding and
polishing is relatively smooth. SEM analysis reveals that the surface
of TC4 alloy (Figures 2A,B), TC4-5Cu alloy (Figure 2B), and TC4-
6Cu alloy (Figures 2B,C)isat and smooth, while the surface of
ETC4 alloy (Figure 2BA), ETC4-5Cu alloy (Figure 2BB), and
ETC4-6Cu alloy (Figure 2BC) was characterized by micro-porous
structures. Most of the visible elements in the present alloys are
titanium (Ti) and a small amount of aluminum (AI) and vanadium
(V). Meanwhile, copper (Cu) can be detected in TC4-5Cu alloy (as
shown in Figures 2B,C)andTC4-6Cualloy(asshowninFigure 2C).
3.2.2 Hydrophilicity
As shown in Figure 3, the contact angle of TC4, TC4-5Cu,
and TC4-6Cu groups and ETC4, ETC4-5Cu, and ETC4-6Cu
groups are 72.14 ± 4.081, 63.23 ± 8.321, and 67.81 ± 3.763 and
68.95 ± 8.536, 64.3 ± 3.972, and 71.07 ± 3.854, respectively. The
contact angle of TC4-5Cu and ETC4-5Cu groups is smaller than
that of the others, but had no statistical signicance (p>0.05).
3.3 Behaviors of RAW 264.7 cells on
different material surfaces
3.3.1 Cell proliferation and cytotoxicity
Figure 4A shows the optical densities of RAW 264.7 cells in
different alloy extracts. With the increase in incubation time,
there is a signicant increase in OD values in each group,
indicating the number of RAW 264.7 cells in each alloy
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TABLE 1 Weight of the samples.
Sample Before etching (g) After etching (g) Weight loss (g) Average
weight loss (g)
TC4 0.8347 0.8152 0.0195 0.0200
0.8343 0.8103 0.0240
0.8354 0.8116 0.0238
0.8351 0.8213 0.0138
TC4-5Cu 0.6280 0.6156 0.0124 0.0236
0.6258 0.6155 0.0103
0.6248 0.5942 0.0306
0.6153 0.5743 0.041
TC4-6Cu 0.8908 0.8797 0.0111 0.0281
0.8874 0.8574 0.0300
0.8861 0.8546 0.0315
0.8847 0.8449 0.0398
FIGURE 2
Surface morphology of alloys: (A) macro images of alloys; (B) SEM images of alloys; (C) EDS analyses of alloys: (A) TC4 alloy and (a)ETC4 alloy;
(B) TC4-5Cu alloy and (b)ETC4-5Cu alloy; (C) TC4-6Cu alloy and (c)ETC4-6Cu alloy.
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extract increased. At the time of 12h, there is no difference
between groups among unetched alloy groups. At the time of
24 h, the absorbance of the TC4-5Cu group is signicantly higher
than that of other groups (p<0.05) among unetched groups; the
absorbance of the ETC4 group is higher than that of the control
group (p<0.05). The relative growth rates (RGR) of RAW
264.7 cells in different alloy extracts are shown in Table 2. The
cell toxicity grade (CTG) is obtained according to the standard
United States Pharmacopeia. From 12 to 24 h of incubation, all
the experimental groups show grades 0 to 1 (no toxicity).
3.3.2 Cell adhesion
Figure 5A shows the adhesion of RAW 264.7 macrophages
cultured on different alloy samples for 24 h. Red uorescence
represents F-actin, and blue uorescence represents the nucleus.
Compared with the TC4-5Cu group, the numbers of RAW
264.7 cells in TC4-6Cu are higher. It could be seen that
compared with all unetched groups and the ETC4 group, the
numbers of RAW 264.7 cells in ETC4-5Cu and ETC4-6Cu
groups are higher, and the areas of the red stained
cytoskeleton of RAW 264.7 cells are also larger, that indicated
that the morphology of RAW 264.7 cells is also more extended
(Figure 5A).
3.3.3 Cell morphology
The morphology of RAW 264.7 cells cultured in all alloy
extracts for 24 h is shown in Figure 6A. Red uorescence stands
for F-actin, and blue uorescence represents the nucleus. The
unactivated M0 phenotype macrophages are spherical-like.
M1 phenotype macrophages are characterized by rounded-like
shapes with extended spread area, while macrophages with
M2 phenotype are usually elongated. After 24 h of
inoculation, RAW 264.7 cells in extracts of all alloy samples
expand pseudopodia and connect, while the cells incubated in
different exact alloy samples show different morphology. Most
RAW 264.7 cells are activated in etched groups, showing M1 or
M2 phenotype, and generally showing elongated spindle shape
with an obvious extension of lamentous pseudopodia. However,
in unetched groups, RAW 264.7 macrophages show round
structure in almost all alloy extracts except TC4-6Cu alloy.
The extension of RAW 264.7 cells is more obvious in the
ETC4-5Cu and ETC4-6Cu groups than in the ETC4 group
(Figure 6A).
3.3.4 M1/M2 phenotype of macrophages
Figure 7A,B shows the expression of M1-type marker
CD86 and M2-type marker CD206 in RAW 264.7 cells
cultured on the surface of different alloy samples for 12 and
24 h. After 12 h culture (Figure 7A), the expression of CD86 in
TC4 alloy, TC4-5Cu alloy, and TC4-6Cu alloy increased by
2.26%, 2.54%, and 4.73%, respectively, and the expression of
FIGURE 3
Contact angle of samples (n= 10, NS: No signicant
difference).
FIGURE 4
Cell proliferation of RAW 264.7 cells cultured in different alloy
extracts for 12 and 24 h (n=5,*p<0.05)
TABLE 2 Relative growth rate (RGR) and cytotoxicity level at different
detection periods.
12 h 24 h
RGR grade RGR grade
Before etching TC4 97.96 ± 7.17 1 96.66 ± 10.90 1
TC4-5Cu 108.70 ± 6.04 0 112.69 ± 3.36 0
TC4-6Cu 102.77 ± 5.40 0 98.99 ± 11.65 0
After etching TC4 113.13 ± 3.11 0 112.26 ± 4.41 1
TC4-5Cu 100.40 ± 8.66 0 106.55 ± 6.32 1
TC4-6Cu 92.72 ± 9.32 1 109.89 ± 7.47 1
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FIGURE 5
Cell adhesion of RAW 264.7 cells cultured on the surface of different alloys for 24 h. (a’–f)is a partial enlargement of the selected area of (AF).
FIGURE 6
Morphology of RAW 264.7 cells cultured in different alloy extracts for 24 h.
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FIGURE 7
Flow cytometry analysis of RAW 264.7 cell surface markers cultured in different alloy extracts for 12 and 24 h (A,C) Expression of CD86 and
CD206 in different alloy extracts for 12 h; (B,D) expression of CD86 and CD206 in different alloy extracts for 24 h (NS: No signicant difference; *p<
0.05; **p<0.01; #, compared with TC4 alloy, p<0.05; and, compared with TC4-5Cu alloy, p<0.05; %, compared with TC4-6Cu alloy, p<0.05)
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CD206 increased by 3.17%, 3.63%, and 4.34% compared with the
control group, respectively. The CD86 marker of ETC4 alloy,
ETC4-5Cu alloy, and ETC4-6Cu alloy increased by 4.53%, 2.24%,
and 6.69%, respectively, and the CD206 marker of ETC4 alloy,
ETC4-5Cu alloy, and ETC4-6Cu alloy increased by 5.09%, 3.89%,
and 4.82% compared with the control group, respectively. After
24 h of culture (Figure 7B), the expression of CD86 in TC4 alloy,
TC4-5Cu alloy, and TC4-6Cu alloy increased by 1.43%, 1.81%,
and 7.69%, and the expression of CD206 increased by 2.93%,
8.67%, and 9.66% compared with the control group, respectively.
The expression of CD86 of ETC4 alloy, ETC4-5Cu alloy, and
ETC4-6Cu alloy increased by 5.1%, 9.34%, and 10.02%, and the
expression of CD206 of ETC4 alloy, ETC4-5Cu alloy, and ETC4-
6Cu alloy increased by 5.72%, 10.62%, and 13.13% compared
with the control group, respectively.
Statistical analysis results display that after 12 h of culture
(Figure 7C), there is no signicant difference in CD86 and
CD206 expression between unetched alloy groups and the
control group, but the expression levels of CD86 and
CD206 of etched alloy groups are signicantly higher than
those of the control group (p<0.05). After 24 h (Figure 7D),
the expression levels of CD86 and CD206 in the ETC4, ETC4-
5Cu, and ETC4-6Cu groups increase compare to the TC4, TC4-
5Cu and TC4-6Cu groups (p<0.05), indicating that acid etching
could increase the expression of CD86 and CD206. The
expression of CD86 in the TC4-6Cu, TC4-5Cu, and
TC4 group decreases gradually (p<0.05), and the expression
of CD206 in the TC4-6Cu group is signicantly higher than that
of the TC4 group (p<0.05), indicating that Cu-bearing titanium
alloy could increase the expression of CD86 and CD206. The
expression of CD86 of ETC4-5Cu and ETC4-6Cu groups is
signicantly higher than that of the ETC4 group (p<0.01),
and the expression of CD206 of ETC4-6Cu alloy is signicantly
higher than that of ETC4 alloy (p<0.05).
3.3.5 Intracellular reactive oxygen species level
Flow cytometry is used to detect the ROS expression of RAW
264.7 cells cultured in different alloy sample extracts for 12 and
24 h (Figure 8). After 12 h, the ROS level in ETC4-5Cu is
signicantly lower than that of the TC4-5Cu group (p<0.05).
FIGURE 8
Flow cytometry analysis of intracellular ROS levels on RAW 264.7 cells in different material extracts for 12 and 24 h. (A,C) Expression of ROS in
different alloy groups for 12 and 24 h; (B,D) quantication of mean uorescence intensity (MFI) of intracellular ROS level in different alloy groups for
12 and 24 h (n = 3, NS: No signi cant difference; *p<0.05; **p<0.01; and, compared with TC4 alloy, p<0.01; ##, compared with TC4-5Cu alloy, p<
0.01; %%, compared with TC4-6Cu alloys, p<0.01)
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The ROS level of TC4-6Cu alloy is signicantly lower than that of
the control group and TC4 group (p<0.01); the ROS levels of
ETC4-5Cu alloy (p<0.01) and ETC4-6Cu alloy (p<0.05) are
signicantly lower than those of the control group. After 24 h, the
ROS level in etched groups is signicantly lower than that of
unetched groups (p<0.01). The ROS level of TC4-6Cu alloy is
signicantly lower than that of TC4 alloy and TC4-5Cu alloy (p<
0.05); the ROS level of the ETC4-6Cu group is signicantly lower
than that of other etched groups (p<0.01), and the ROS level of
ETC4-5Cu alloy is signicantly lower than that of ETC4 alloy
(p<0.05).
4 Discussion
The 3D printing technology used in this study is selective
laser melting (SLM), which creates solid models by adding
powder layer by layer. Laser power, scanning speed, scanning
spacing, and single layer thickness are the major parameters in
the process of selective laser melting. Different laser processing
parameters were compared, and the optimal parameters were
selected in this study: laser power of 260 W, scanning distance of
45 µm, and laser spot diameter of 70 µm. Physical and chemical
characteristics of implant material surfaces, such as surface
morphology and hydrophilicity, can play a crucial role in the
process of bone integration (Smeets et al., 2016). Copper, as an
essential trace mineral for human beings, has been proven to
have excellent antibacterial properties and has been widely used
in titanium alloyrelated studies (Fan et al., 2022). In this study,
TC4-5Cu alloy and TC4-6Cu alloy were made by adding
different content of copper into TC4 alloy using 3D printing
technology, and the printed alloys were treated by a traditional
acid etching technique.
The ratio of HF, HNO
3,
and H
2
O used in acid etching
treatment and the acid etching duration all affect the surface
morphology of TC4 alloy, and the etching duration is particularly
important. It is reported that hydrophilic surfaces with lower
contact angles are more conducive to cell attachment and
proliferation (Rupp et al., 2017). It was found that the
hydrophilicity was not signicantly affected by acid etching
(Figure 3). Therefore, the differences in cell growth and
attachment between groups were not related to their
hydrophilicity. In addition to hydrophilicity, the release of
metal ions may also affect cell proliferation and adhesion (Lai
et al., 2020). Acid etching may generate increased immersion of
copper ions that could affect cell proliferation and adhesion. In a
previous study, it was shown that etched TC4 alloy surface in HF-
HNO3 solution may lead to selective dissolution of α-phase and
selective enrichment of β-phase on the alloy surface (Sittig et al.,
1999). In addition, acid etching treatment could also clean
pollution particles on the surface of TC4 alloy (Yi et al., 2022).
This study prepared alloy extracts from TC4, TC4-5Cu, and
TC4-6Cu alloys ETC4, ETC4-5Cu, and ETC4-6Cu alloys
according to ISO10993-12. The copper element in the alloy
was gradually dissolved and released into the medium, thus
affecting the biological behavior of macrophages in the
material alloy extracts. However, high doses of copper can
lead to formation of free radicals and induce cytotoxicity by
causing chromosome and DNA damage (Sharma et al., 2021). In
this study, the CCK-8 test showed that Cu-bearing titanium
alloys (TC4-5Cu alloy and TC4-6Cu alloy) had no effect on the
viability of RAW 264.7 cells compared with the TC4 alloy, and all
Cu-bearing TC4 alloy samples had no cytotoxicity (Figure 4).
Therefore, Cu-bearing titanium alloys could be used as long-term
implantation materials. In addition, acid etching treatment did
not affect the cell viability of various alloys and proven to have no
cytotoxicity.
In this study, the adhesion of RAW 264.7 cells on the surface
of alloy materials was observed by staining. The results showed
that RAW 264.7 cells could attach and extend well on the surface
of all alloy samples after 24 h incubation (Figure 5). Meanwhile,
compared with the surface of the TC4 alloy, the surface of Cu-
bearing titanium alloys (TC4-5Cu alloy and TC4-6Cu alloy) is
benecial for the adhesion of RAW 264.7 macrophages
(Figure 5). The surface of TC4-6Cu alloy shows better cell
adhesion ability than that of TC4-5Cu alloy. The possible
reasons are as follows: 1) with the increase of copper content,
the porosity of TC4 alloy also increases, and the surface area of
Cu-bearing titanium alloy and cells also increases
correspondingly, which improves the ability of cell adhesion;
2) Protein adsorption is the prerequisite for cell adhesion to
biomaterials (Yang et al., 2021), and the hydrophilic surface of
Cu-bearing titanium alloy may promote protein adsorption. The
possible reasons are as follows: 1) with the increase of copper
content, the porosity of TC4 alloy also increases, and the surface
area of Cu-bearing titanium alloy and cells also increases
correspondingly, which improves the ability of cell adhesion;
2) protein adsorption is the prerequisite for cell adhesion to
biomaterials (Yang et al., 2021), and the hydrophilic surface of
Cu-bearing titanium alloy may promote protein adsorption. The
etched alloy samples showed stronger cell adhesion to RAW
264.7 cells than that of unetched alloy samples (Figure 5). The
reasons may be as follows: 1) acid etching treatment improves the
surface morphology of the alloy, and micro-porous structures,
which were observed by SEM (Figure 2B), could increase the
surface area of material alloys and immerse more copper ions to
promote cell adhesion; 2) acid etching treatment can clean the
pollution particles on the surface of TC4 alloy, which is more
conducive to the growth of cells.
Confocal microscopy was used to observe the cell
morphology of RAW 264.7 cells in different alloy extracts.
Macrophages can polarize into different phenotypes to
perform different functions. The shapes of polarized
macrophages are different. M1-type macrophages are generally
fried egg-shapedand M2-type macrophages were mostly long
spindle-shaped (McWhorter et al., 2013). In this study, the RAW
Frontiers in Materials frontiersin.org10
Yan et al. 10.3389/fmats.2022.941311
264.7 cells cultured in etched sample extracts for 24 h are
activated and show the structural features of the
M2 phenotype, including elongated spindle shape and
lamentous pseudopodia; while the cell structures of unetched
groups are mostly round. (Figure 6). In addition, RAW 264.7 cells
in etched Cu-bearing titanium alloy groups (ETC4-5Cu alloy and
ETC4-6Cu alloy) have more obvious lamentous pseudopod
extension and interconnection. (Figure 6).
The observed macrophage surface marker proteins were
detected by ow cytometry in different groups. The results
showed that the expression of M1-type surface marker
CD86 and M2-type surface marker CD206 was increased in
Cu-bearing titanium alloy groups, indicating that copper in Cu-
containing titanium alloys could promote the polarization of
macrophages. Compared with the same alloy before and after
acid etching, CD86 and CD206 in etched groups were
signicantly higher than those in unetched groups, indicating
that etching treatment could promote the polarization of
macrophages (Figure 7).
The production of reactive oxygen species (ROS) is the
core of inammatory response in the early implantation
stage. Oxidative stress occurs when ROS levels exceed the
antioxidant defenses in cells, leading to irreversible
intracellular damage (Schieber and Chandel, 2014). In the
production process of 3D printed medical titanium alloy,
partially melted or un-melted powder may remain on the
surface of the materials and may lead to residual particle
pollution and ROS generation in the adhesion cells. In this
study, RAW 264.7 cells were cultured in the alloy sample
extracts to detect the ROS production level. The results
showed that at the time of 24 h, ROS levels in all etched
alloy groups were signicantly reduced compared with those
in unetched alloy groups (Figure 8). The possible reason is
that the acid etching treatment cleans the residual particles
on the surface of the alloy. The ROS level in ETC4-5Cu and
ETC4-6Cu alloy groups is obviously lower than that of other
groups. The ROS level in the TC4-6Cu alloy group is lower
than that of TC4-5Cu, and the ROS level in the ETC4-6Cu
alloy group is lower than that of the ETC4-5Cu alloy
group. With the increase in copper content, the ROS levels
decreased. These results suggest that ROS production may be
closely related to phenotype switching of macrophages, and
ROS production may promote the M1-type macrophage
polarization.
Macrophages are considered to be one of the earliest cells
reaching the surface of the implant. The appropriate
inammatory reaction between the macrophage and implant
is benecial for the success of implantation (Franz et al., 2011).
Macrophages polarize into M1 type (classical activation type) or
M2 type (alternative activation type), expressing different
functional programs according to different micro-
environmental signals (Linares et al., 2016).
M1 macrophages mainly exist in the early stage of
inammation and act as pro-inammatory cells to spread
the inammatory response to downstream immune cells.
M2 macrophages mainly contribute to stopping
inammation and promoting tissue repair and wound
healing. It was reported that M1-type macrophages are
dominant in the failed tissues surrounding implants, while
M2-type macrophages are dominant in the normal tissues
surrounding implants by Tazzyman et al. (Tazzyman et al.,
2013). In this study, the etched alloy surfaces with lower ROS
levels could promote macrophages to polarize to the M2-type
phenotype, thus contributing to the increase in the success rate
of implant implantation. This may be caused by acid etching
treatment which makes the surface of the material form a
microporous structure and more copper-element can be
precipitated from the copper-bearing titanium alloy. The
copper element can activate macrophages and tends to
polarize macrophages to M2-phenotype.
However, the polarization of macrophages on different Cu-
bearing alloy surfaces is not only closely related to ROS levels but
also affected by the levels of many inammatory factors.
Therefore, the relevant inammatory factors on RNA and
protein levels need further exploration in the following
experiments.
5 Conclusion
All samples had no toxicity to RAW 264.7 cells. With the
increase of copper content, the adhesion ability of cells on the
surface of the material was enhanced. Adding copper to
TC4 alloys could activate the polarization of macrophages on
the surface of TC4 alloy and reduce the ROS level. Surface
treatment by acid etching had no effect on the hydrophilicity
of the alloy surface. Acid etching treatment also could enhance
cell adhesion, activate macrophage polarization, and reduce ROS
levels. The in vitro experiments of this study indicated that acid-
etched 3D printed copper-bearing titanium alloy could promote
the adhesion and polarization of macrophages and provide a
promising implant material for the treatment of peri-implant
inammation.
Data availability statement
The original contributions presented in the study are
included in the article/Supplementary Material; further
inquiries can be directed to the corresponding authors.
Author contributions
BL and HK contributed to the conception and design of the
study. QW designed and prepared the materials. JY performed
Frontiers in Materials frontiersin.org11
Yan et al. 10.3389/fmats.2022.941311
the experiments and wrote the manuscript. HK helped perform
the analysis with constructive discussion. WH polished the
language of the article. All authors have read and approved
the nal version of the manuscript.
Funding
This study was nancially supported by the Natural
Science Foundation Project of Liaoning Province (Nos.
2020-MS-150 and 2018225059) and Guangxi Key
Laboratory of the Rehabilitation and Reconstruction for
Oral and Maxillofacial Research Funded Project (No.
GXKLRROM2107).
Conict of interest
The authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
be construed as a potential conict of interest.
Publishers note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their afliated organizations,
or those of the publisher, the editors, and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher.
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3D printed titanium implants have gained substantial attention in the medical fields of bone tissue and dental implants. Surface modification of titanium implants such as TiO2 nanotubes (TNT) make up for the interaction between the surface of titanium implants and the surrounding tissues by providing nanoporous structures and hydrophilic surfaces. In this study, a 3D printed Cu-bearing Ti6Al4V (TC4) alloy with micro/nano-topographical was employed as material model to explore the role played by Cu²⁺ and nanostructure in the physical-chemical properties of the material, osteoblast toxicity. The role of Cu²⁺ and nanostructure on macrophage proliferation, polarization and oxidative stress without and with lipopolysaccharide (LPS) stimulation was also evaluated. Surface characterizations showed TNT significantly increased the roughness and surface hydrophilicity of TC4 alloy. TNT-TC4-5Cu exhibited excellent corrosion properties in the absence and presence of LPS. The in vitro tests with MC3T3-E1 cells demonstrated that the TNT-TC4-5Cu alloys showed the better cytocompatibility. Moreover, TNT-TC4-5Cu alloy can inhibit macrophage polarization and decreased ROS generation without and with LPS stimulation, thereby promoting the biocompatibility. In summary, the TNT-TC4-5Cu alloy promoted the proliferation and adhesion of osteoblasts and inhibited the inflammatory response of macrophages, which has great potential to benefit the future development of orthopedic applications.
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In the field of orthopaedics, inflammation-modulatory biomaterials are receiving increasing attentions due to their abilities to regulate innate immune response and mediate wound healing. In the current work, a Cu-containing micro/nano-topographical bio-ceramic surface (Cu-Hier-Ti surface) was employed as material model to explore the role played by Cu²⁺ release or material surface in regulating macrophage polarization as well as macrophage-mediated osteogenic and bactericidal effect. A Cu-free micro-topographical surface (Micro-Ti surface) generated by micro-arc oxidation was employed as control. The results showed that Cu²⁺ supplemented directly into the culture medium or released from Cu-Hier-Ti surface could polarize macrophages to pro-inflammatory M1 phenotype by activating Cu-transport signaling (copper transporter 1 (CTR1) and ATP7A) in macrophages, while the material characteristics exhibited anti-inflammatory effect to some extent by regulating integrin (α5, αM, β1 and β2) and TLR (TLR-3, TLR-4, Myd88 and Ticam-1/2) signaling. Macrophages grown on Cu-Hier-Ti surface or treated by Cu²⁺ could create a favorable inflammatory microenvironment for osteoblast-like SaOS-2 cell proliferation and differentiation. Moreover, Cu-Hier-Ti surface promoted macrophage capacity to engulf and kill bacteria, even though it did not show direct bactericidal effect against Staphylococcus aureus. In vivo results showed that Cu-Hier-Ti surface could lead to promoted osteointegraion and enhanced expression levels of M1 surface marker CD11c, growth factor BMP-6 and osteogenic makers including osteocalcin (OCN) and Runx-2 at the biomaterial/bone tissue interface in a rat model. The results indicate that Cu could be employed as a promising inflammation-modulatory agent to activate macrophages for enhanced osteogenic and bactericidal effect. Statement of significance The next generation of bone biomaterials should be active to regulate the local inflammatory environment such that it favors bone regeneration. For the design and development of Cu-containing inflammation-modulatory biomaterials, it is of great importance to recognize the exact role played by Cu²⁺ release or material surface characteristics. So far, relatively little is known about the regulatory role of Cu²⁺ or micro/nano-topographical surface on macrophages. The results in the current work suggest that Cu²⁺ release and material surface characteristics of Cu-containing micro/nano-topographical coating could activate distinct signaling pathways in macrophages. The activated M1 macrophages exhibited stimulatory effect on osteoblast maturation and enhanced bactericidal capacity against Staphylococcus aureus. This study might provide new thoughts for the development of multi-functional Cu-containing biomaterials.
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In a diabetic milieu high levels of reactive oxygen species (ROS) are induced. This contributes to the vascular complications of diabetes. Recent studies have shown that ROS formation is exacerbated in diabetic monocytes and macrophages due to a glycolytic metabolic shift. Macrophages are important players in the progression of diabetes and promote inflammation through the release of pro-inflammatory cytokines and proteases. Because ROS is an important mediator for the activation of pro-inflammatory signaling pathways, obesity and hyperglycemia-induced ROS production may favor induction of M1-like pro-inflammatory macrophages during diabetes onset and progression. ROS induces MAPK, STAT1, STAT6 and NFκB signaling, and interferes with macrophage differentiation via epigenetic (re)programming. Therefore, a comprehensive understanding of the impact of ROS on macrophage phenotype and function is needed in order to improve treatment of diabetes and its vascular complications. In the current comprehensive review, we dissect the role of ROS in macrophage polarization, and analyze how ROS production links metabolism and inflammation in diabetes and its complications. Finally, we discuss the contribution of ROS to the crosstalk between macrophages and endothelial cells in diabetic complications.