Research Article

Cite This: ACS Appl. Mater. Interfaces 2019, 11, 6809−6819 www.acsami.org

Mechanical Strength, Biodegradation, and in Vitro and in Vivo

Biocompatibility of Zn Biomaterials
Donghui Zhu,*,† Irsalan Cockerill,† Yingchao Su,† Zhaoxiang Zhang,‡ Jiayin Fu,§ Kee-Won Lee,∥
Jun Ma,† Chuka Okpokwasili,⊥ Liping Tang,⊥ Yufeng Zheng,# Yi-Xian Qin,¶ and Yadong Wang§
†
Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207, United States
‡
Department of Plastic Surgery and ∥Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261,
United States
§
Nancy E and Peter C. Meinig School of Biomedical Engineering, Cornell University, New York 14853, United States
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⊥
Department of Bioengineering, University of Texas at Arlington, Arlington, Texas 76010, United States
#
Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China
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¶
Department of Biomedical Engineering, Stony Brook University, New York 11794, United States
*
S Supporting Information

ABSTRACT: Zn-based biomaterials have emerged as promising new types of bioresorbable metallics applicable to orthopedic
devices, cardiovascular stents, and other medical applications recently. Compared to other degradable metallic biomaterials (i.e.,
Mg- or Fe-based), Zn biomaterials have a more appropriate corrosion rate without hydrogen gas evolution. Here, we evaluated
the potential of Zn-based metallics as medical implants, both in vitro and in vivo, alongside a standard benchmark Mg alloy,
AZ31. The mechanical properties of the pure Zn were not strong enough but were significantly enhanced (microhardness > 70
kg/mm2, strength > 220 MPa, elongation > 15%) after alloying with Sr or Mg (1.5 at. %), surpassing the minimal design criteria
for load-bearing device applications. The corrosion rate of Zn-based biomaterials was about 0.4 mm/year, significantly slower
than that of AZ31. The measured cell viability and proliferation of three different human primary cells fared better for Zn-based
biomaterials than AZ31 using both direct and indirect culture methods. Platelet adhesion and activation on Zn-based materials
were minimal, significantly less than on AZ31. The hemolysis ratio of red cells (<0.5%) after incubation with Zn-based materials
was also well below the ISO standard of 5%. Moreover, Zn-based biomaterials promoted stem cell differentiation to induce the
extracellular matrix mineralization process. In addition, in vivo animal testing using subcutaneous, bone, and vascular
implantations revealed that the acute toxicity and immune response of Zn-based biomaterials were minimal/moderate,
comparable to that of AZ31. No extensive cell death and foreign body reactions were observed. Taken together, Zn-based
biomaterials may have a great potential as promising candidates for medical implants.
KEYWORDS: Zn alloy, biocompatibility, orthopedic, cardiovascular, corrosion, mechanical property

■ INTRODUCTION
Biodegradable metals are metals that can corrode progressively
in the human body to full dissolution while completing the
objective to support tissue regeneration without implant
residues.1,2 Ideally, the released corrosion products should
induce minimal host response and toxicity, thus initial design
should avoid using components known to induce these
effects.2 Therefore, the bulk composition of biodegradable
metals should be composed of essential metallic elements,
which degrade at desirable rates and can be easily metabolized
by the human body through absorption or excretion.1−5
Primary bioresorbable metals include iron (Fe), magnesium
Received: November 23, 2018
Accepted: January 29, 2019
Published: January 29, 2019

© 2019 American Chemical Society 6809 DOI: 10.1021/acsami.8b20634

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(Mg), and zinc (Zn). The past few decades have concentrated
on Fe- and Mg-based biomaterials, but the success of such
metals is still slightly limited. Recent studies revealed that Fe
might be an inappropriate material because of its slower
degradation compared to the healing rate and the long-term
retention of a voluminous chemically stable iron oxide.6−8
Therefore, Fe can hardly be considered a real “bioresorbable”
metal. Conversely, Mg degrades too fast, usually within 1−4
months, far under the desired time frame of at least 4−6
months for orthopedic or stent applications.9,10 Also, the
significant hydrogen gas evolution and pH change associated
with Mg degradation in the local environment are another big
concern in clinical applications. Moreover, current typical Mg
alloys with slightly enhanced corrosion resistance (e.g., AZ-
and WE-series alloys) for medical implants usually contain
aluminum, zirconium, and/or rare earth elements, which are
potentially toxic.11−14 While still showing some promises, the
design of a new Mg-based alloy as an ideal cardiovascular or
orthopedic implant material presents a significant metallurgical
challenge.
Metallic Zn is a physiologically relevant metal but has not
yet been fully considered as a bioresorbable medical implant.
Compared to titanium and stainless steels, their physical and
mechanical properties are much more in line with human
bone.15,16 The typical drawbacks associated with Fe- and Mg-
based alloys such as unsuitable corrosion rate and lack of
ductility are usually not applied to Zn-based alloys.17
Furthermore, Zn2+ is the second most copious element to
humans, confined primarily to muscles and bones.18−21
Importantly, over 600 enzymes rely on Zn2+ for proper
orientation and function.20 However, research on Zn
biomaterials as medical implants is still limited,22−35 with
most of them focused on in vitro analysis of mechanical and
corrosion properties.
Pure Zn itself is not strong enough for load-bearing medical
applications. To improve its mechanical strength, Zn needs to
be alloyed with other elements to form Zn-based alloys.
Although some recent studies showed that alloying with
elements including aluminum, rare earth, neodymium, and
yttrium could significantly enhance Zn alloys’ mechanical
properties,1,2,15 most of these alloying elements are not
desirable because of their potential toxicity. Therefore, group
IIA nutrient elements of the periodic table such as Mg, Ca, and
Sr are preferred because they are important mineral supple-
ments for human health.2,36,37
Taken together, these findings inspired us to logically
investigate Zn-based biomaterials as potential alternatives, or
even replacements, for medical implants based on their
biodegradation, mechanical, and biocompatibility properties.
Research studies on this promising biodegradable material are
still largely lacking. Using a typical Mg alloy, AZ31, as our
comparative benchmark, we examined Zn-based metallics as
potentially better bioresorbable metals for medical applica-
tions, including their mechanical strength, biodegradation,
cytocompatibility, stem cell differentiation, and acute inter-
actions (up to 1 month) with connective, bone, and vascular
tissues in animal models.
■
MATERIALS AND METHODS
Material Preparation. Pure Zn (99.9998%) and extruded
magnesium alloy AZ31 (Mg96/Al3/Zn1) were obtained from
Goodfellow. Zn binary alloys, Zn−Sr and Zn−Mg with 1.5% (atomic
%) of alloying elements Sr or Mg, were selected because the plasticity
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and formability of Zn alloys with 1.5% Mg or Sr were found to be
significantly improved when compared to the pure Zn in our pilot
studies (data not shown). They were prepared from pure Zn
(99.99%), pure Sr (99.99%), or Mg (99.99%) ingots in a high-purity
graphite crucible as described before.22 A gas mixture of 1 vol % SF6/
CO2 balance was used to prevent burning and oxidation. After being
held at 630 °C for 0.5 h, the melt pool was transferred to a 150 °C
preheated steel mold. As-cast binary alloys were subsequently hot-
rolled or extruded to increase the mechanical properties. Rolled sheets
and the extruded cylinders were then cut into standard testing
specimens for mechanical testing (Vickers hardness and tensile and
uniaxial compression testing) as described before.22 In addition, disk
samples (Φ 10 × 1.5 mm) including AZ31, pure Zn, Zn−Sr, and Zn−
Mg alloy specimens were prepared for corrosion tests, hemolysis,
platelet adhesion, and cell experiments. Wires of various diameters
and 10 mm lengths were prepared through extrusion-drawing
methods for implantation into mouse or rat animal models. For all
biological testing, specimens were ground up to 4000 grit SiC to
smooth out the surface, cleaned ultrasonically for 15 min each in
acetone, ethanol, and distilled water, disinfected with 70% (v/v)
ethanol for 30 min, and UV-sterilized for 2 h.22
Mechanical Tests. Tension and uniaxial compression tests were
performed according to ASTM-E8M-09 and ASTM E9-89a (2000)
standards as described before.22 Vickers hardness was evaluated with a
100 g applied load and a 15 s loading time. Vickers hardness (Hv) was
calculated according to the formula: Hv = 1.8544P/d2, where Hv is in
units of kilogram per square millimeter, the indenter load (P) is in
kilogram, and the indenter diagonal length (d) is in millimeter.22
Immersion Tests. According to ASTM-G31, samples were soaked
in simulated body fluid (SBF) solution as described before with slight
changes.38,39 At different time intervals, a precalibrated pH meter was
used to assess medium pH. No medium was changed or replenished
during the process of immersion. Then samples were removed from
SBF solution after up to 8 weeks. Specimens were then examined by
scanning electron microscopy (SEM) to characterize surface
morphological changes. To remove corrosion products from the
surface, specimens were washed with a solution of chromic acid (200
g/L CrO3 and 10 g/L AgNO3) for a duration of 10 min. Samples were
then taken out and dried at room temperature.40,41 Sample weights
were determined prior to and after removal of corrosion products.
The corrosion rate (C) was calculated according to C = Δm/ρAt,
where C is in units of millimeter per year, Δm is the mass loss, ρ is the
material density, A is the starting surface area, and t is the time of
immersion.
Cell Culture. Human coronary artery endothelial cells (HCAECs),
human osteoblast cells (HOBs), and bone marrow-derived human
mesenchymal stem cells (hMSCs) were cultured as described
before.19,20,38,39,42−46 HCAECs were purchased from ScienCell and
cultured with endothelial cell medium, including 5% fetal bovine
serum (FBS) and supplemented with 1% endothelial cell growth
supplement and 1% penicillin/streptomycin. HOBs were obtained
from Thermo Fisher Scientific and cultured in 10% FBS Dulbecco’s
modified Eagle’s medium/F12 supplemented with 1% penicillin/
streptomycin. hMSCs were cultured with nucleotide-free 10% FBS α-
minimum essential medium supplemented with 1% penicillin/
streptomycin. Standard culture conditions of 37 °C, 5% CO2, and
95% relative humidity were applied, and cell medium was replaced
every 2−3 days. HCAECs and HOBs at passages 4−6 and hMSCs at
passages 3−5 were used for experiments. ISO 10993-5/12 culture
standard was applied to prepare media extracts from the samples.
Cell Viability. Cell viability was evaluated through the 3-[4, 5-
dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT) assay
(Thermo Fisher Scientific, US) as described before.19,20,38,39,42−46
Briefly, cells were seeded into 96-well plates at 10 000 cells/well and
incubated for 24 h to enable attachment. Specific extract with different
concentrations then replaced the cell medium, and cells were cultured
for up to 5 days (n = 8). Following this, culture medium with 10%
mM MTT solution replaced the extract medium. Sodium dodecyl
sulfate−HCl (100 μL) was added to the culture wells after 4 h of
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incubation time. After another 4 h, absorbance of each well was read
at 570 nm. Cells cultured with normal medium were used as control.
Cell Proliferation. To evaluate the cytotoxicity of our materials
from different perspectives, the CyQUANT direct cell proliferation
assay (Thermo Fisher Scientific, US) was used to determine the
proliferation rate of different cells as described before.19,20,42 Cells
were allowed to attach for 24 h at a density of 10 000 cells/well. Cell
medium was swapped for extract medium, and cells were cultured for
up to 5 days (n = 8). 1× detergent reagent replaced extract solution,
and cells were again cultured for 1 h, prior to reading sample plate
fluorescence (F) from bottom at 480/520 nm.
Cell Adhesion. Cell adhesion analysis was performed as described
before.19,20,39,42,45,46 Briefly, cells were seeded onto the sample
surfaces, and cells were incubated under standard conditions for 5 h
(n = 6). Subsequently, culture medium was removed, and the cells
were rinsed with Dulbecco’s phosphate-buffered saline (DPBS). Cells
were stained by Calcein AM prior to fixation using 4%
paraformaldehyde. Adhered cells were visualized by an EVOS FL
Cell Imaging System fluorescence microscope (AMG, US) and
further studied by ImageJ. Cell density and retention ratio were
confirmed through a minimum of 10 different fields.
Hemolysis Test. Hemolysis test was performed as described
before.39 Briefly, fresh healthy human blood (Cedarlane Lab, US) was
diluted with 1× DPBS at 4:5 volume ratio. Sample pieces (n = 6)
were placed in 10 mL of 1× DPBS along with 0.2 mL diluted blood.
Each tube was then incubated at 37 °C for 1 h before centrifuging at
3000 rpm. Absorbance of the supernatants was read at 545 nm.
Positive and negative controls included diluted blood with 10 mL
deionized water and 10 mL 1× DPBS, respectively.
Alkaline Phosphatase Activity. The alkaline phosphatase (ALP)
activity assay kit (Thermo Fisher Scientific, US) was used to measure
ALP activity by following the manufacturer’s instructions.32 Briefly,
stem cells with a seeding density of 6000 cells/cm2 were cultured in a
cell differentiation medium atop material surfaces for a total of 7 days
with medium changed every 3 days (n = 8). Then, cell lysate was
collected, and total protein was determined by the bicinchoninic acid
assay. Next, supernatants were mixed with a StemTAG AP Activity
Assay Substrate for 30 min at 37 °C. Addition of 1× Stop Solution
completed the reaction, and absorbance was read at 405 nm,
normalized by the total protein, which was used to determine the ALP
activity.
Alizarin Red Activity. hMSC osteogenic differentiation was
assessed via the Alizarin Red quantification assay by following the
manufacturer’s instructions (Thermo Fisher Scientific, US).32 Briefly,
stem cells were cultured for 20 days on material surfaces in plates (n =
8). Cells were lysed from the material surface using 10% acetic acid,
and lysates were heated for 10 min at 85 °C. Cell lysates were then
cooled for 5 min on ice, prior to 15 min centrifugation at 20 000g.
Then, 200 μL supernatant was collected and mixed with the same
volume of ammonium hydroxide to neutralize the acid. ARS
concentration was obtained from the supernatant absorbance at 405
nm and a preprepared standard curve.
RT-PCR for the mRNA Measurement. Collagen I and
osteopontin gene expression level also indicate the osteodifferentia-
tion of the hMSCs, which is examined by CFX96 Touch RT-PCR
Detection System (Bio-Rad, US) as described before.32 Total RNA
was extracted [RNeasy Mini Kit (Qiagen, US)] and calculated with a
Nanodrop spectrophotometer. RNA was reverse-transcribed in a
thermo cycler (T100, Bio-Rad, US) using the RT2 First Strand Kit
(Qiagen, US). β-Actin mRNA level served as the endogenous control
and reference. Final mRNA expression data of target protein were
obtained using the 2 − ΔΔCt method with Bio-Rad CFX Manager 3.1
software.
In Vivo Subcutaneous Implantation. The University of Texas
at Arlington’s IACUC approved all procedures in accordance with
NIH care and use of laboratory animal guidelines. All metallic
implants (AZ31, Zn, Zn−Sr, and Zn−Mg) and sham-operated control
received dorsal midline incisions following anesthetization in C57
mice (Jackson Labs; n = 6) as previously described.47,48 Briefly, a
subcutaneous incision was made in each mouse, and two metallic
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implants were placed laterally approximately 15 mm on either side of
the incision. Incisions were sealed with surgical clips, and mice were
monitored for up to 4 weeks in housing for irritation until explanted.
In Vivo Bone Implantation. All procedures were approved by
the IACUC at Peking University according to NIH guidelines for the
care and use of laboratory animals. We adopted a rat femur condyle
model (n = 12 for each different implant) as described before.49
Briefly, after being anesthetized, male young Sprague-Dawley rat (2−3
months) with an average of 200 g was immobilized, and a longitudinal
incision approximately 10 mm in length alongside the lateral extensor
mechanism and close to the knee joint was made. A bone defect,
measuring 5 mm depth and 2 mm in diameter, was created through
the distal bone epiphysis gap. Zn implant was then placed through rat
femoral condyles along with sham-operated control. The wound was
closed carefully, and the rats were rested and housed in standard
conditions. The rats were sacrificed after 1 month, and 10% formalin
buffer was used to fix femoral condyles for histomorphometric
analysis.
Histomorphometric Analysis of the Bone Tissue. We
processed the explants for bone tissue staining as described before.49
Briefly, explants were fixed, ethanol was dehydrated, xylene was
cleared, and methyl methacrylate was embedded. Samples were cut
along their vertical axes. After grinding and polishing, section samples
were stained with van Gieson Picrofuchsin. Microscopy (Olympus
CKX41, Japan) images were utilized to image and examine the
specimens.
In Vivo Vascular Implantation. Cornell University’s IACUC
approved all procedures in accordance with NIH guidelines for the
care and use of laboratory animals. Sprague-Dawley young male rats
(2−3 months, weight = 225−250 g, Charles River Laboratories,
Boston, MA) were used for alloy wire implantation (n = 6 for each
different implant). Isoflurane induction (5%) and 2% maintenance
were used to anesthetize the rats. An abdominal midline incision 20
mm in length exposed the abdominal aorta, which was then separated
from the inferior vena cavas. Both proximal and distal parts of the
aorta were clamped with microvascular movable clips. Wires made of
AZ31, Zn, Zn−Sr, and Zn−Mg in the size of 0.25 mm diameter were
cut as 10 mm length, rinsed with acetone, and disinfected for 30 min
with 70% (v/v) ethanol and 2 h with UV prior to implantation. To
contact the wire with circulating arterial blood, both ends were
inserted into the lumen throughout the proximal and distal parts of
the arterial wall by puncturing the arterial adventitia and leading the
wire in, followed by a second puncturing in the arterial
adventitia.50−52 Microvascular movable clips were removed from the
aorta, and the instant blood flow was checked with a flow Doppler for
5, 10, and 30 min. The surgical site was closed with 5−0 Dacron
sutures. No anticoagulation or antiplatelet treatments were adminis-
trated pre- and postoperatively. Antibiotic (Baytril, 20 mg/kg) and
analgesic (Buprenex, 0.02 mg/kg) medications were given until 72 h
postoperatively.
Vascular Explantation and Histological Analysis. At 1 month
postimplantation, rats were anesthetized, and a midline incision
exposed the abdominal aorta. The aorta including Zn wire was taken
out from surrounding tissues for analysis. The explanted wire/aorta
was rinsed with sterile phosphate-buffered saline, fixed for 1 h in 10%
neutral formalin, and soaked for 24 h at 4 °C in 30% sucrose solution.
The whole wire/aorta was embedded vertically into the optimal
cutting temperature (OCT) compound (Sakura Finetek, Torrance,
CA), snap-frozen at −80 °C, and cryosectioned as 10 μm thickness.
Hematoxylin and eosin (H&E) staining of the slides was done on the
vascular tissue surrounding the wire.
Hematoxylin and Eosin. At 1 month postimplantation,
surrounding tissues including implants were excised for histological
evaluation and immunofluorescence staining. Specifically, biomaterial-
induced inflammatory reactions were measured by quantifying
macrophages, neutrophils, mast cells, and fibrocytes at the implant−
tissue interface as described in our works.47,53−57 Surrounding tissues
and alloys were excised for frozen sectioning by embedding in OCT.
Then, cross sections (7 μm) were made, and cell infiltration of the
different tissue layers was visualized by H&E staining. Interface
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Figure 1. Mechanical properties of Zn, AZ31, Zn−Sr, and Zn−Mg alloys: (A) microhardness; (B) tensile properties; and (C) elongation. Scale bar
= 2.5 mm (aP < 0.05, bP < 0.01, cP < 0.001).
Figure 2. Immersion test of AZ31 and Zn materials in a time course up to 30 days. (A) Pictures of AZ31 (left) and Zn (right) after up to 30 days of
immersion in SBF; (B) cumulative Mg ion released from AZ31; (C) cumulative Zn ion released from Zn; (D) change of pH value in solution; and
(E) calculated corrosion rate of Zn and AZ31 from immersion testing (cP < 0.001).
response quantification was carried out using ImageJ as reported
previously.47,48 Briefly, images taken on the skin side of the tissue−
biomaterial interface are shown as the multiple count averages of
H&E stains.
Statistical Analysis. All data were presented as mean ± standard
deviation. Assays were repeated 3 times autonomously with a
minimum of three replicates. One-way or two-way analysis of
variance followed by Tukey’s post-hoc test or two-tailed Student’s t-
test, as appropriate, was used to analyze the statistical significance. P <
0.05 was considered as statistically significant.
■ RESULTS
Mechanical Strength. Mechanical properties are signifi-
cant factors for load-bearing bioresorbable metals (e.g., bone
plates/screws and vascular stents); thus, they should have
enough strength comparable to the local tissues as for
mechanical performance. The mechanical properties of pure
Zn are poor. To meet the needs of the implant, strengthening
strategies include alloying and hot-working deformation, which
were tested here.39 After alloying with Sr or Mg, the
microhardness of Zn increased from 40 to over 70 kg/mm2
(Figure 1A). The yield strength (YS) and ultimate tensile
strength (UTS) were significantly improved from less than 50
to over 220 MPa after alloying (Figure 1B). A similar trend was
observed for elongation, which increased from ∼5 to over 15%
after alloying with Sr or Mg (Figure 1C). It is noteworthy that
Zn−Sr and Zn−Mg had higher microhardness and elongation
than that of benchmark control, AZ31, while they all had
similar YS and UTS. Such data demonstrate that through
alloying, Zn-based biomaterials can be developed with
sufficient strength, rendering them as appropriate load-bearing
biomedical implants.
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Corrosion Behavior. Ideally, the corrosion or biodegra-
dation rate of implants should meet the pace of tissue healing.
We then examined the corrosion behavior of the materials
based on immersion testing. Figure 2A shows the surface
morphology of samples soaked in SBF up to 30 days. The Zn
samples retained flat surfaces after 30 days, whereas the
surfaces of AZ31 were covered with corrosion products.
Cumulative Mg ion released from the alloy AZ31 increased
linearly over the immersion period, whereas cumulative Zn ion
released from Zn materials reached a plateau after ∼20 days,
indicating slower corrosion after initial stage for Zn (Figures
2B,C and S1). Immersion medium pH change was recorded
(Figures 2D and S1). Degradation of AZ31 induced significant
pH increase compared to control and Zn over time. However,
degradation of Zn did not induce significant pH change. Figure
2E shows the calculated corrosion rate of AZ31 and Zn-based
materials from immersion testing. The corrosion rate of Zn-
based materials was approximately 0.4 mm/year significantly
slower than that of AZ31.
Cytocompatibility by Indirect Culture. Primary
HCAECs, HOBs, and hMSCs were cultured with a
concentration gradient of extract solutions from Zn and
AZ31 materials over the course of 1 (Figure 3), 3 (Figure S2),
and 5 days (Figure S3). Increasing extract concentration
reduced HCAEC viability and proliferation in an overall trend
(Figure 3A,B). However, cell viability and proliferation
reduced much more significantly for AZ31 than Zn at higher
concentrations (P < 0.05). This is probably due to the
increasing levels of ions (i.e., Mg2+, Al3+, and Zn2+) and pH in
the extract solution of AZ31. Moreover, different cells have
different sensitivities or tolerances to extracellular ion
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Figure 4. Direct culture of primary HCAECs, HOBs, and hMSCs on

the surfaces of AZ31 and Zn plates for 1−5 days with quantitative cell
adhesion density measurements: (A) cell attachment on AZ31; (B)
quantitative measurement of cell density on AZ31; (C) cell
attachment on Zn; and (D) quantitative measurement of cell density
on Zn (aP < 0.05).

Figure 3. Culture of primary HCAECs, HOBs, and hMSCs with

extracts of AZ31 and Zn materials for 1 day. Cell viability (A,C,E) and
cell proliferation (B,D,F) were measured separately for these three cell
types, respectively (aP < 0.05, bP < 0.01, cP < 0.001).

concentrations.19,20,42 Interestingly, the cell viability and

proliferation of HOBs and hMSCs did not decrease after
treatment with Zn or AZ31 medium extracts (Figure 3C−F).
When the extract concentrations were lower, they promoted
the cell growth of HOBs and hMSCs (P < 0.05). Among the
three cell types, HCAECs were the most sensitive to the
medium extracts and had the least tolerance. AZ31 was the
most toxic to HCAECs as most HCAECs died after 3−5 days
of culture. On the contrary, no significant cell toxicity of AZ31
and Zn was observed for HOBs and hMSCs at the same
culture conditions (Figures 3, S2, and S3).
Cytocompatibility by Direct Culture. We also per-
formed in vitro cytocompatibility testing by direct culture of
primary HCAECs, HOBs, and hMSCs on surfaces of Zn and
AZ31 plates for up to 5 days. For HCAECs, there were hardly
any viable cells (green) on AZ31, whereas there were
numerous viable cells on Zn (Figure 4A,C). The density of
HCAECs on Zn increased significantly with increasing culture
time, indicating fast growth and proliferation on the Zn surface
(Figure 4B,D). For both HOBs and hMSCs, confluent cell
densities on Zn were achieved over 5 day time course (Figure
4B,D). Cell densities on AZ31 were initially low, but over time Figure 5. Hemocompatibility analysis of AZ31 and Zn materials. (A)
SEM images of platelet adhesion on the sample surface; (B)
they increased significantly, reaching subconfluency after 5
quantitative amount of the adhered platelet; and (C) hemolysis of
days (Figure 4A,C). Similar cytocompatibility results were AZ31 and Zn-based materials (aP < 0.05, bP < 0.01).
observed for Zn−Sr and Zn−Mg materials (Figure S4).
Hemocompatibility. We carried out the blood compati-
bility testing as shown in Figure 5. The adhered platelet mostly of round shape, lacking pseudopodia spreading (Figures
morphology on plates of AZ31 and Zn-based metallics was 5A and S5), indicating no/minimal platelet activation. The
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number of platelets on the top of Zn-based materials was far found that tissue responses to Zn-based materials were
less than on AZ31 (Figure 5B). These results indicate superb comparable to those of AZ31 (Figures 8 and S6). Only a
antithrombotic and antiplatelet adhesion properties. Our
studies also showed relatively low (<0.4%) (Figure 5C)
hemolysis rates on Zn-based materials, which are far below
ISO 10993-4:2002 5% safe value standards, indicating that Zn
alloys will not cause serious hemolysis.
In Vitro Mineralization. It has been reported that Zn
materials promote bone regeneration in vivo.49 Thus, we
examined the in vitro mineralization process at the cell−
material interface by ALP and Alizarin Red staining.
Interestingly, Zn-based materials stimulated significantly
more ALP activity of hMSCs as compared to controls when
the differentiation medium was applied (Figure 6A). In

Figure 6. Osteogenic differentiation of hMSCs as measured by ALP

(A) and Alizarin Red (B) with the treatment of extracts of AZ31 and
Zn-based materials (10% as referred to Figure 3) (aP < 0.05, bP < Figure 8. H&E staining of tissues surrounding different implants for 4
0.01). weeks. (A) Representative images of H&E and (B) quantitative cell
numbers around the implants (* implantation area).
addition, Zn-based materials induced significantly more calcific
deposition and activity of hMSCs evidenced by the Alizarin
Red level staining by the levels of Alizarin Red staining small number of inflammatory cells were found at the interface
compared to controls (Figure 6B). Moreover, osteopontin and between the tissue and alloys. The population of macrophages
collagen I mRNA levels were significantly higher as compared around the Zn-based implants was also comparable to that of
to control, and Zn-based materials had even slightly more AZ31, as measured by CD11b staining (Figures 9 and S7).
mRNA expression of these genes compared to AZ31 (Figure
7A,B), consistent with a preceding study.32

Figure 7. Osteogenic differentiation of hMSCs after different

treatments as measured by mRNA levels of collagen I (Col I) and
osteopontin (OPN). (A) Fold change of the Col I mRNA level as
compared to control. (B) Fold change of the OPN mRNA level as
compared to control (aP < 0.05).

In Vivo Immune Response. We first evaluated the in vivo
tissue biocompatibility of our Zn biomaterials and AZ31
control using a subcutaneous implantation model in mice. The
extent of tissue responses to the alloy implant was assessed
based on the extent of inflammatory responses and fibrotic
tissue formation at the implant−host tissue interface. We
Figure 9. CD11b staining of the tissues surrounding different
implants after 2 weeks. (A) Representative images of CD11b positive
cells and (B) quantitative cell numbers around the implants. The red
arrowhead points to a representative CD11b positive cell, and the
green arrowhead points to a representative CD11b negative cell (*
implantation area).

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ACS Applied Materials & Interfaces
Figure 10. Histological characterization of the vascular tissue in a rat abdominal aorta implantation model. (A) Explanted pure Zn wire/aorta at
day 30 postimplantation. (B) Top: H&E staining of the complete cross section in different regions and bottom: partial magnification of the box
shown in top images. (C) H&E staining of the healthy aorta control. Scale bar = 200 μm (w: wire).
In Vivo Vasculature−Implant Interaction. Next, we
assessed in vivo vascular cell/tissue responses of pure Zn wires
by implanting them in rat abdominal aorta for 1 month (Figure
S8). In this study, the explanted tissues appeared to be normal
and comparable to sham-operated control, indicating no
embolism and thrombosis in the animals tested (Figure
10A). H&E staining of the explants showed the foreign
response to the wires. There were some inflammatory cells and
tissues in the adventitial side, which underwent fibrotic
responses, resulting in layers of infiltrated inflammatory cells
and extracellular matrix (ECM) deposition (Figure 10B). No
extensive inflammation was observed. Same experiments were
performed for AZ31 and Zn alloys, and similar results were
observed (data not shown).
In Vivo Bone−Implant Interaction. In addition, we
tested if Zn implant will promote osteointegration, osteocon-
duction, and/or osteoinduction using a rat femoral condyle
implantation model. New bone formation was observed
surrounding the Zn implant via van Gieson staining with an
abundance of osteocytes in the newly formed bone (Figure
11). There were also some fibrotic and collagenous tissues
Figure 11. Histological characterization of bone tissue sections at
implant sites with Van Gieson staining 1 month postimplantation of
Zn. The yellow rectangles correspond to the magnified bone−implant
interface. The green triangle indicates a newly formed bone (*
implantation area).
between the implants and newly formed bones. Moreover,
stretching outward from the biomaterial, expanding bone mass
was observed, which indicated an osteogenic property of Zn.
Similar results were observed for Zn−Sr and Zn−Mg alloys
(data not shown), consistent with previous studies.22,58
■
DISCUSSION
Biodegradable metals are designed to provide a temporary
support after implantation, with the expectation of being
completely reabsorbed during the tissue healing process. There
are three main biodegradable metals Mg, Zn, and Fe. Zn
biodegradable metals emerge as a new generation of metallic
implants largely for orthopedic and cardiovascular stent
applications lately. Using a typical Mg alloy, AZ31, as a
comparative standard control, we demonstrated that Zn
6815
Research Article
biomaterials after alloying generally have good mechanical
strength suitable for load-bearing implants such as bone
fixation implants and cardiovascular stents, better corrosion
rates matching to the tissue healing pace, and good
cytocompatibility and tissue compatibility with minimal
toxicity and immune responses in animal models.
Alloying with Sr or Mg Enhances the Mechanical
Strength of Zn with Minimal Effect on Degradation and
Biocompatibility. As expected, alloying with Mg or Sr
elements can significantly enhance the mechanical properties
of Zn materials to surpass the minimal design requirements of
load-bearing medical implants, consistent with previous
studies.23,26,49 Mechanical characteristics of some Zn and
various Zn alloys in comparison to that of Mg and natural bone
are summarized in Table 1. Similar to the Mg and Mg alloys,
alloying increased the mechanical properties of Zn, including
mechanical strength and elongation. The mechanical strength
of two Zn alloys in this study is comparable to that of AZ31,
while their elongations are higher than that of the AZ31 alloy.
Although Zn−Li alloys seem to have the strongest strength,
our results are still in line with previous studies.22,59
Pure metals usually have lower strength compared to those
alloyed with other metals, so does Zn. After alloying, the
formation of intermetallics and different metallic phases is
thought to reduce the movement of dislocations within the
metallic structure. The reasons that Sr or Mg alloying can make
Zn stronger are that two basically structural changes may
happen. The first is solid solution strengthening that involves
the addition of other metallic elements dissolving in the parent
lattice and causing distortions because of the difference in
atom size between the parent metal and the solute metal. In
addition, adding elements that have no or partial solubility in
the parent metal will result in the appearance of a second phase
distributed throughout the crystal or between crystals, which
may raise or reduce the strength of an alloy. In this study, a
trace amount of Sr or Mg was used to avoid potential overdose
toxicity despite their natural existence in human body as a
supplemental nutrition. Therefore, attention should be paid to
the selection of the alloying elements. Preference should be
given to nutrient or, at least, nontoxic elements to avoid
potential add-on toxicity from the alloying additions. Although
significant mechanical strengthening was observed after
alloying, there were no obvious changes in corrosion rate
and biocompatibility compared to pure Zn. Data also showed
that there was no significant difference between Zn−Sr and
Zn−Mg for all the mechanical strength, corrosion, and toxicity
tested so far.
DOI: 10.1021/acsami.8b20634
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ACS Applied Materials & Interfaces Research Article

Table 1. Summary of Mechanical Properties of Various Implant Materials and Natural Bone
materials UTS (MPa) YS (MPa) elongation (%) compressive YS (MPa)
natural bone 100−15063 1−363 130−1809
pure Mg 85 (as-cast) 20 (as-cast) 10−163 65−1009
165 (hot-rolled)3 110 (hot-rolled)3
pure Zn49,64 18 (as-cast) 10 (as-cast) 0.2 (as-cast) 105 (extruded)
50 (hot-rolled) 30 (hot-rolled) 5.5 (hot-rolled)
60−110 (extruded) 33−50 (extruded) 3.5−60 (extruded)
Zn1X (Mg, Ca, Sr)31,49,64,65 150−185 (as-cast) 120−130 (as-cast) 1−2 (as-cast) 280−340 (extruded)
220−250 (hot-rolled) 188−205 (hot-rolled) 12−20 (hot-rolled)
240−270 (extruded) 200−220 (extruded) 8−11 (extruded)
Zn3Mg31,49,64,65 30 (as-cast) 291 (extruded) 0.2−0.4 (as-cast)
399 (extruded) 1 (extruded)
Zn4Li34 450 (hot-rolled) 420 (hot-rolled) 14 (hot-rolled)
Zn(1−3)Al35,64 220−240 (hot-rolled) 134−210 (hot-rolled) 22−32 (hot-rolled)
AZ31a 280 245 13
pure Zna 50 30 6
Zn-1.5Mga 285 240 17
Zn-1.5Sra 250 220 22
a
Data from this study.

Zn-Based Metallics Have a More Suitable Degrada-
tion Rate Than That of Mg and Fe. The major problems
with Mg-based metals are the too fast degradation rate, the
hydrogen gas evolution, and insufficient mechanical strength
for some load-bearing applications. The major drawbacks with
Fe-based metals are the prolonged degradation time and the
hard-to-clear degradation products retained in the local tissues.
The degradation rate of Zn is between that of Mg and Fe
because of standard electrode potential (−0.76 V/SCE) of Zn
falling in the middle of Mg (−2.37 V/SCE) and Fe (−0.44 V/
SCE).15,31 This explains why we observed a moderate and
similar degradation rate of pure Zn and two Zn alloys. It might
also be due to the formation of similar passive degradation
films for all Zn-based metallics.
Systemic Toxicity of Zn Implants Is Minimal. Similar to
Mg or Fe, Zn is an essential trace element. Zn deficiency may
cause significant disorders, and implant-derived Zn ions
released inside the body may be beneficial if not overdosed.60
Naturally, overdose is always a concern as Zn’s tolerable upper
intake level (UL) is 15−40 mg/day, significantly lower than
that of Mg (300−400 mg).61 Thanks to its much slower
corrosion rate than that of Mg, the accumulated Zn ion inside
the body is anticipated to be below the UL value. Therefore,
the risk of systemic Zn toxicity associated with Zn alloys is
negligible based on the following observations.2 Here is our
rationale. Assuming that a typical Zn implant weighs ∼250 mg
of pure metal with a time frame of 12 months for a total
degradation, the expected daily dose of Zn will be <1 mg/day.
As the recommended daily value of Zn is about 10 mg/day,2
the amount of Zn released from the degradation of the Zn
alloy implant is only 10% of the recommended daily intake for
adults, which is far below its recommended daily intake value.
Thus, Zn implant systemic toxicity appears not a concern, even
with multiple devices placed in the same patient. The local
toxicity should also be minimal as demonstrated by the data
from previous studies22 and our studies here, possibly due to
the swift transport and clearance of metallic ions, including the
Zn ion, from the tissue to peripheral blood flow,19,20 thus
prohibiting its enrichment and cytotoxicity in the vicinity of
the implant.
6816
In Vitro and in Vivo Biocompatibility of Zn-Based
Implants Is Generally Good. Biocompatibility is another
important factor for degradable biomaterials besides mechan-
ical and corrosion properties. Overall, Zn materials exhibit
comparable, sometimes better hemocompatibility and cyto-
compatibility than the comparative control AZ31. This could
be due to its slow degradation rate, less local pH change, and
minimal hydrogen gas evolution. Testing conditions, param-
eters, and cell types could be other factors as well. In animal
models, the toxicity and immune responses of Zn materials
were also minimal and comparable to AZ31. It is notable that
Zn alloys promoted significant bone regeneration in a mouse
model as reported recently,49 consistent with our in vitro
mineralization and in vivo data. Formation of a new bone was
visualized by histological analysis at week 4. Some fibrotic and
collagenous tissues between the new bone tissue and materials
were also observed. This could be explained by the high Zn ion
concentration in the vicinity of implants, which may hinder the
formation of a new bone. Therefore, fibrotic tissues may form
during bone tissue healing to cause a delayed osseointegration.
Zn degradation products include zinc oxide, zinc hydroxide,
and Ca/P compounds, in addition to zinc and hydroxide ions.
As an essential element for various biological functions, Zn
may promote new bone formation as demonstrated by
enhanced mineralization of the ECM, differentiation of
hMSCs, and enhanced expression of genes related to bone
remodelings such as ALP, collagen I, and osteopontin. Zn may
also inhibit osteoclast differentiation. On the other hand,
extreme Zn ion release locally may induce adverse effects on
cells, leading to potential bone resorption in vivo. Therefore,
large concentration of Zn ions released from rapidly
degradable implants should be carefully controlled.
Our studies here generally focused on in vitro and acute in
vivo evaluations, and a long-term experiment is needed to fully
understand the potential of Zn-based biomaterials for different
biomedical applications. Nonetheless, there is no doubt that
Zn-based biomaterials may have a greater potential for medical
implants.62
DOI: 10.1021/acsami.8b20634
ACS Appl. Mater. Interfaces 2019, 11, 6809−6819
ACS Applied Materials & Interfaces
■ CONCLUSIONS
Using a typical Mg alloy, AZ31, as a comparative benchmark
control, we examined the potential of some Zn biomaterials as
medical implants, both in vitro and in vivo. We found that Zn
biomaterials after alloying exhibited sufficient mechanical
strength for load-bearing device applications. The corrosion
rate of Zn biomaterials is significantly slower than that of
AZ31. Degradation of AZ31 also induced significant pH
increase over time but not for Zn-based materials. The
measured blood compatibility, cell viability, and proliferation
of three different human primary cells fared better for Zn
biomaterials than AZ31. Zn biomaterials also promoted stem
cell differentiation to induce the ECM mineralization process.
Moreover, in vivo animal testing revealed that the toxicity and
immune response of Zn biomaterials were minimal/moderate,
comparable to that of AZ31. No extensive cell death and
foreign body reactions were observed. Thus, Zn biomaterials
are demonstrated to be promising bioresorbable metal
contenders for cardiovascular and orthopedic applications.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.8b20634.
Immersion tests of Zn−Sr and Zn−Mg materials; cell
viability and proliferation of primary HCAECs, HOBs,
and hMSCs with extracts of AZ31 and Zn materials for 3
and 5 days, respectively; interactions between cells and
Zn−Mg or Zn−Sr; hemocompatibility analysis of Zn−Sr
and Zn−Mg; H&E staining of tissues surrounding Zn−
Sr and Zn−Mg; CD11b staining of the tissues
surrounding Zn−Sr and Zn−Mg implants; and
implantation of the Zn wire in the rat abdominal aorta
(PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: Donghui.Zhu@unt.edu. Phone: 940-369-8707.
ORCID
Donghui Zhu: 0000-0002-3057-1889
Yufeng Zheng: 0000-0002-7402-9979
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health
(grant number R01HL140562). The content is solely the
responsibility of the authors and does not necessarily represent
the official views of the National Institutes of Health.
■
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