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OPEN A promising biodegradable

magnesium alloy suitable for
clinical vascular stent application
received: 17 October 2016 LinMao1,2,3, Lishen4, JiahuiChen4, XiaoboZhang5, MinsukKwak3, YuWu3, RongFan3,
accepted: 14 March 2017 LeiZhang2, JiaPei2, GuangyinYuan2, ChengliSong1, JunboGe4 & WenjiangDing2
Published: 11 April 2017
We report a Mg alloy Mg-2.2Nd-0.1Zn-0.4Zr (wt.%, denoted as JDBM-2) showing great potential in
clinical vascular stent application by integrating the advantages of traditional medical stainless steel
and polymer. This alloy exhibits high yield strength and elongation of 2766MPa and 34.33.4%
respectively. The JDBM-2 with a stable degradation surface results in a highly homogeneous
degradation mechanism and long-term structural and mechanical durability. In vitro cytotoxicity test
of the Mg extract via human vascular endothelial cells (HUVECs) indicates that the corrosion products
are well tolerated by the tested cells and potentially negligible toxic effect on arterial vessel walls. This
alloy also exhibits compromised foreign body response (FBR) determined by human peripheral blood
derived macrophage adhesion, foreign body giant cell (FBGC) formation and inflammatory cytokine
and chemokine secretion. Finally, vascular stents manufactured from the JDBM-2 were implanted into
rabbits for long-term evaluation. The results confirm excellent tissue compatibility and up to 6-month
structural and mechanical integrity of the stent in vivo. Thus, the JDBM-2 stent with up to 6-month
structural and mechanical integrity and excellent tissue compatibility represents a major breakthrough
in this field and a promising alternative to traditional medical stainless steel and polymer for the clinical
application.

Coronary heart disease (CHD), the number one killer of both men and women worldwide, counts for 34 and
41% of all death in the United States1 and in China2, respectively. Since the idea of placing stents in blood vessels
was first proposed by Dotter et al. in 19643, coronary stenting has evolved into the most common catheter-based
treatment of coronary heart disease due to its lower restenosis rate than percutaneous transluminal coronary
angioplasty (PTCA)4.
In the past decades, cardiovascular stents have been manufactured using two material classes: metals and poly-
mers. Of these two, polymers for load-bearing application are relatively weak and less rigid compared with metals.
Moreover, polymer stents are also associated with an increased risk of late stent thrombosis (ST)5. The conven-
tional metallic implants based on stainless steel68, Co-Cr alloy911, Ni-Ti alloy1214 and Ta15 are the commonly
used materials for coronary artery reconstruction because of their high mechanical properties and excellent
corrosion resistance. However, the long-term host foreign body response (FBR) to permanent stent implanta-
tion often results in restenosis or lumen encroachment, brought about by the recruitment of inflammatory and
smooth muscle cells to the intima with subsequent proliferation and extracellular matrix (ECM) deposition16,17.
Hence, metallic materials, which can be absorbed and incorporated into the tissues, appear to provide an
approach to avoid the long-term endothelial dysfunction and foreign body reaction. Iron and Mg alloys are pos-
sible candidates for use in biomedical implants. Whereas the corrosion period of iron is too long18, Mg-based
implants appear to offer appropriate degradation periods being closer to the clinical requirements19. Moreover,
Mg alloys show excellent anti-platelet deposition and low thrombogenicity, which makes it particularly promising

1
Shanghai Institute for Minimally Invasive Therapy, School of Medical Instrument and Food Engineering, University
of Shanghai for Science and Technology, Shanghai, 200093, China. 2National Engineering Research Center of Light
Alloys Net Forming and State Key Laboratory of Metal Matrix Composite, Shanghai Jiao Tong University, Shanghai,
200240, China. 3Department of Biomedical Engineering, Yale University, New Haven, CT 06511, USA. 4Shanghai
Institute of Cardiovascular Diseases, Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai,
200032, China. 5School of Materials Science and Engineering, Nanjing Institute of Technology, Nanjing, 211167,
China. Correspondence and requests for materials should be addressed to G.Y.Y. (email: gyyuan@sjtu.edu.cn) or
C.L.S. (email: csong@usst.edu.cn)

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Figure 1. Microstructures of the JDBM-2 alloy in (a) T4, (b) once extrusion and (c) double extrusion
conditions. (d) The inset in (c) showing SEM image of double extruded JDBM-2 dispersed with nano particles
in grain interiors and boundaries.

Samples YS (MPa) UTS (MPa) Elongation (%)

T4 66 3 181 5 10.2 1.3
Once extrusion 204 5 247 4 20.6 1.6
Double extrusion 276 6 309 6 34.3 3.4

Table 1. Mechanical properties of the JDBM-2 alloy in T4, once and double extrusion conditions.

for cardiovascular intervention20,21. More importantly, Mg is one of the trace elements existing in the human
body, the corrosion product of Mg alloy generated by the electrochemical reaction Mg+ 2H2O Mg (OH)2+ H2
can be absorbed or excreted by the surrounding tissues and metabolic system22. Previous studies have showed
great promising of Mg alloys for biomedical application since the first Mg stent was implanted into a preterm baby
in 2004 by Zartner et al.19,23,24.
Despite recent advances in the improvement of corrosion properties via a variety of technical methods, Mg
alloys for the clinical application of biodegradable cardiovascular stent is still challenged by the short-term sup-
port of less than 6-month durability due to the rapid galvanic-corrosion, which is undesirable to cause com-
plete failure of the medical device before the tissue has healed completely. Here, we report a promising Mg alloy
JDBM-2 with great potential by integrating the advantages of medical stainless steel and biodegradable polymer
for vascular stent application. The cardiovascular stent manufactured by this Mg alloy with up to 6-month struc-
tural and mechanical integrity and excellent tissue compatibility achieved through controllable homogeneous
degradation mechanism represents a major advancement in the evolution of stent and also opens opportunities
to improve the long-term clinical outcome of percutaneous coronary intervention.

Results
Microstructures and Mechanical properties. Figure1ac shows the microstructures of the JDBM-2 in
solution treatment (T4), once extrusion and double extrusion conditions. The microstructure of the JDBM-2 after
double extrusion becomes much finer and more homogeneous than those in T4 and once extrusion conditions
due to complete dynamic recrystallization in hot processing. Fine dispersed nano particles (precipitated phases)
which contain 3.0wt% Nd and 1.6wt% Zr precipitates in grain interiors and boundaries of the JDBM-2 after
double extrusion (Fig.1d, Figure 1s).
The mechanical properties of the JDBM-2 in T4, once and double extrusion conditions are shown in Table1.
After double extrusion, the ultimate tensile strength (UTS), yield strength (YS) and elongation of solution treated
JDBM-2 are increased by 306%, 70 and 209% respectively (typical stress-strain curves shown in Fig. 2s). The nano
particles act as barriers for the movement of dislocations during sliping and contribute to the yeild strength. On
the other hand, the refined homogeneous microstructure after double extrusion is favorable for the plastic defor-
mation. The improvement in elongation of JDBM-2 after double extrusion could be attributed to the activated
non-basal dislocations in fine-grain Mg alloy with a better grain boundary compatibility effect25.

In vitro degradation in artificial plasma. The in vitro degradation experiment was carried out by immers-
ing the double extruded JDBM-2 alloy in artificial plasma for 120h and the corrosion surface was cleaned by a
standard chromium trioxide (CrO3) solution to remove the products, as shown in Fig.2. Comparison was made
with a conventional Mg alloy AZ31 (Fig.2a). The corrosion of the JDBM-2 spreads across the surface, which
results in very uniform and smooth corrosion morphology (Fig.2b). In contrast, pitting corrosion is extensively
observed on AZ31Mg alloy due to the inherent micro-galvanic corrosion. The corrosion rate of the JDBM-2
(T4) measured by mass loss and hydrogen evolution is reduced by 25% from 0.49 to 0.37mm/year after double
extrusion (Fig.2c), revealing an enhanced corrosion resistance of the JDBM-2 achieved through a fine and homo-
geneous microstructure. The H2 evolution volume (Fig.2d) generated by the double extruded JDBM-2 is much
less than that generated by the alloy in T4 condition during the investigated period. The result is found to be good
agreement with the in vitro corrosion rate calculated by mass loss. Figure2e shows the cyclic polarization curve
for double extruded JDBM-2 after immersion in artificial plasma for 1h. The corrosion potential of the reverse

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Figure 2. In vitro degradation of Mg alloy in artificial plasma. Macro corrosion surfaces of (a) conventional
AZ31 alloy and (b) double extruded JDBM-2 alloy after exposing to physiological medium for 120h and
cleaning the corrosion products. (c) The corrosion rates of T4 and double extruded (denoted as DE) JDBM-2
alloy calculated by mass loss and H2 evolution. (d) The volume of H2 evolutions of T4 and double extruded
JDBM-2 alloy in artificial plasma for every 1-day degradation. (e) Cyclic polarization curve of double extruded
JDBM-2 alloy after immersed in artificial plasma for 1hour to reach a steady state. The arrows with the sequence
from one to four indicate the scanning direction of the polarization curve. Note the lower corrosion potential of
the forward scan which represents the polarization behavior of the non-corroded area in the Mg alloy.

scan (1.68V) is greater than that of the forward scan (1.71V), indicating the corrosion in the corroded area
on JDBM-2 alloy is likely to be suppressed by the non-corroded area, and the non-corroded area is more easily
attacked by the corrosion due to its lower corrosion potential. As a result, the corrosion of JDBM-2 alloy spreads
across the surface and leads to more homogeneous corrosion.

In vitro cytotoxicity test. To explore cellular response to the corrosion products, we carried out stud-
ies by in vitro cytotoxicity assay of HUVECs exposed to the JDBM-2 (double extrusion condition) extract.
Immunofluorescence images stained for cytoskeleton and nuclei show that HUVECs appear normal and healthy
with a typical morphology of cobblestone in the Mg extract compared with those cultured in normal cell culture
medium (Fig.3a). Fig.3b illustrates HUVECs viability expressed as a percentage of negative control after 1, 3
and 5 days of culture in diluted extract media with 10 and 50% concentrations, respectively. The cytotoxicity test
shows slight negative effect on HUVECs viability (75~99%, Grade 1) in the first day incubation, while a signif-
icant recovery of cell viability (100%, Grade 0) for all tests was detected on days 3 and 5. The BrdU incopora-
tion of HUVECs (Fig.3c) cultured in diluted JDBM-2 extracts shows slight negative effect on cell proliferation
potential in the first day incubation, and then rises to more than 80% of normal cell proliferation potential for all
tests with different extracts on days 2 and 3, reflecting a rapid restoration of normal cell behavior. The cytotoxic-
ity evaluation via human endothelial cells demonstrates that the JDBM-2 extract possesses favorable biological
compatible with human endothelial cell.

In vitro morphology of macrophages and inflammatory secretion detection. To investigate the

effects of implanted materials on programming of macrophages into a continuum of diverse functional states,
primary macrophages from human peripheral blood were cultured directly on the JDBM-2 substrate. 316L stain-
less steel served as negative control. A metal-based cell culture system (detailed fabrication processes and meth-
ods were illustrated in Fig.4) was used for macrophages/FBGCs culture and supernatant collection for protein
analysis. After periods of 12h and 72h, macrophage population distributes uniformly on 316L stainless steel
while it accumulates on degradable JDBM-2 substrate, suggesting that the JDBM-2 is unfavourable for mac-
rophage adhesion and growth and consequently leads to lower density of macrophages participating in FBR.
Immunofluorescence examination shows that macrophages spread to bring their membranes into close contact,
which enables macrophages to come into a cytoskeletal rearrangement and fusion-competent status for mem-
brane merging to form multinucleated cells (Fig.5a,b). Quantitative analysis confirms that decreased levels of

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Figure 3. Cytocompatibility test of corrosion products in vitro on human endothelial cells. (a) Morphologies
of HUVACs in negative control and Mg extract with10 and 50% dilution (from left to right) for 24h. The cells were
fixed and stained for cytoskeleton (Pholloidin: green) and nuclei (DRAQ5: purple). (b) HUVEC viability shown as
the percentage of viable cells in the control after 1, 3 and 5 days of culture in the diluted extracts. (c) Proliferation
ratio of HUVECs cultured in diluted Mg extracts for 1, 2, 3 days as compared to negative control.

Figure 4. Schematic showing the experimental setup for the cell culture system and collected
cytokine detection, respectively. The cell culture and cytokine detection system are fabricated from
polydimethylsiloxane (PDMS) with individual hole size of 75mm on top of the substrate and microarray
anti-body glass. Antibody microarrays were manufactured using a pin-spotting technique for assaying a
panel of proteins produced by macrophages/FBGCs. An immunosandwich-based assay was used to detect the
secretion footprint of human macrophages/FBGCs seeded on metal substrates.

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Figure 5. In vitro macrophage fusion and inflammatory cytokine and chemokine detection.
Immunofluorescence images showing the morphology of macrophages/FBGCs cultured on (a) JDBM-2 and
(b) 316L stainless steel for 12h, 72h and 144h (from left to right). The cells were fixed and stained for
cytoskeleton (Pholloidin 532nm: green) and nuclei (DRAQ5 650nm: purple). Multinuclear giant cells
(arrowheads) were observed on both 316L stainless steel and JDBM-2 substrates due to the foreign body
reaction. Cytokine and chemokine production of macrophages/FBGCs cultured on JDBM-2 and 316L stainless
steel for (c) 24h and (d) 72h were determined by ELISA.

macrophage fusion are driven by the JDBM-2 substrate compared with 316L stainless steel (not shown here).
Results of this investigation reveal that a compromised FBR is induced by the JDBM-2. The degradation of the
JDBM-2 leads to an alkalization effect with an increase in pH value of the local microenvironment, which exerts
an important effect on macrophage viability26 and function in terms of adhesion and spread. Furthermore, the
homeostasis of the surface with gradually increased corrosion products caused by the degradation of the JDBM-2
may diminish the absorption of proteins, which serve as extracellular matrix (ECM) for macrophage adhesion
via several adhesion ligand-receptors interacting with those adsorbed proteins27. FBGC formation is a classic
identifying feature of unresolved inflammation arising from the presence of implants and other foreign bodies28.
During the foreign body reaction in vivo, monocytes are recruited to the site of implant insertion, and undergo
maturation to macrophages which may subsequently fuse to form FBGCs. Macrophages lose most of their plas-
ticity and mobility after they become FBGCs, at the same time, they acquire the capacity to release more effective
cytokines, growth factors, and other bioactive agents to degrade foreign bodies and modulate the function of
other cell types which participate in the integration and failure of implants28,29. Results of this study reveal that
the temporary exist of the self-dissolved JDBM-2 stent would take advantage to avoid the long-term FBR in vivo
after the fulfillment of its functionality.
Inflammatory cytokine and chemokine production are distinctly influenced by the surface physico-chemical
properties of biomaterials (Fig.5c,d). Tumor necrosis factor-alpha (TNF-) is detected at lower level expressed
by adherent macrophages/FBGCs cultured on the JDBM-2 possibly due to a lower cell density and less FBGC
formation. TNF-expression is consistent with the initiation of an acute inflammatory response30 and a key reg-
ulator to immediate tissue response by inducing other cytokines and growth factors and consequently influencing
endothelial function and leukocyte transmigration31. Therefore these findings indicate that a compromised acute
inflammatory is initiated by the JDBM-2, suggesting limited stimulation of this material for immune cells. The
in vitro investigations enable us to isolate the interaction between biomaterials and macrophages involving in

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Figure 6.The in vivo aortic angiography showing no acute and late thrombogenesis as well as in-stent
restenosis in the (a) JDBM-2 and (c) 316L stainless steel stent after stenting for 1 month, 2 months, 4 months
and 6 months (from left to right). The corresponding follow-up IVUS images illustrating the longitudinal
reconstruction of the abdominal aorta after the (b) JDBM-2 and (d) 316L stainless steel stent implantation.
Note increased lumen patency and vessel size at different implantation period with nearly complete absence of
neointimal hyperplasia.

FBR and provide perspective for future in vivo assessments. While the investigation identifies the importance of
biomaterial in modulating macrophage behavior and inflammatory process in vitro, the mechanisms by which
macrophages preferentially accumulate and acquire a proinflammatory phenotype need to be further deciphered.

In vivo angiography and IVUS results. We carried out in vivo angiography and the follow-up IVUS to
investigate the safety and efficacy of the biodegradable JDBM-2 stent in rabbit abdominal aorta (Fig.6a,b). The
angiography images show no acute and late thrombogenesis, as well as serious in-stent restenosis in the JDBM-2
stents. The results are similar to those observed in the control group (316L stainless steel), as shown in Fig.6c,
demonstrating that the biodegradable JDBM-2 stent is safe and efficient in vivo (Fig.6a). The follow-up IVUS was
used to evaluate the expansion level, initial hyperplasia degree and the occurrences of thrombosis in different
implantation periods, as shown in (Fig.6b). The JDBM-2 stents are completely expanded and well apposed to the
vessel wall with no sign of elastic recoil and fracture, reflecting excellent radial strength and compliance of the
stent. In period of 1 month implantation, a thin layer of endothelium appears on the surface of the stent struts,
with no significant difference compared with the results achieved in 316L stainless steel stents (Fig.6d). The deg-
radation period of the JDBM-2 stent in rabbit model can reach up to 6-month (arrows indicate the presence of the

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Figure 7. HE staining images of the stented arteries showing compromised foreign body reaction and
neointimal coverage on the JDBM-2 struts in the period of (a) 1 month, (b) 2 months, (c) 4 months and (d) 6
months implantation. The inset in each image showing detailed view of the stented artery. The white and black
boxes indicating that the support period of the JDBM-2 scaffold can achieve up to 6 months.

stent), which is consistent with the result calculated from the corrosion rate in vitro and proves that the JDBM-2
stent offers appropriate degradation rate in vivo. These results are highly striking and the major improvement in
long-term mechanical durability and excellent tissue compatibility is related to the homogeneous degradation
mechanism and slow corrosion rate, which makes JDBM-2 a promising Mg-based biomaterial for manufacturing
biodegradable stent.

HE staining and in stent endothelialization. We further carried out Hematoxylin-Eosin (HE) staining
to evaluate in vivo FBR to the biodegradable JDBM-2 stent (Fig.7). Histologic examination reveals that a few
inflammatory cells accumulate in the vicinity of the stent and a thin layer of neo-endothelium covers the surface
of the struts after 1 month implantation (Fig.7a). A continuous endothelium layer lines on the stented arteries
with compromised inflammatory reaction in the extended periods of implantation. However, mild in-stent inti-
mal hyperplasia is observed in the late periods of 4 and 6 months (Fig.7c,d), and the inflammation basically sub-
sides at the end of the implantation. Meanwhile, the struts of the JDBM-2 stent degrade continuously from 2 to 4
months and maintain their shapes till the end of the study. Combining the results of the in vivo aortic angiography
and the corresponding follow-up IVUS that no early and late in-stent restenosis observed in the JDBM-2 stents,
we conclude that slight inflammatory response and mild in-stent intimal hyperplasia is induced by the JDBM-2
stent during the implantation periods.
We also investigated the distal segment of the stented artery to identify in-stent endothelialization via scan-
ning electron microscope (SEM). Results of this study indicate that complete neointimal coverage on the struts
post stenting is mainly finished in the initial 4 weeks and few inflammatory cells are visible in the endothelialized
stents (Fig.8a,b). In summary, the JDBM-2 stent causes mild inflammatory reaction in the early periods, inhibits
in-stent restenosis after stenting and maintains its structural and mechanical integraty till 6 months implanta-
tion. The attenuation of the accumulation of inflammatory cells and the minor inflammatory reaction in rabbit
abdominal aortic arteries can be reasonably attributed to the re-endothelialization and neointimal coverage on
the stent struts, which has been demonstrated as the main underlying mechanism of late stent thrombosis (ST).

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Figure 8. The SEM images showing neointimal coverage of the (a) JDBM-2 and (b) 316L stainless steel stent
after stenting for 1 month, 2 months, 4 months and 6 months (from left to right). Comparable strut coverage is
evident on the two scaffolds after 4 months implantation.

Figure 9. Schematic diagram of satisfactory stent material required for clinical application and limitations
of current biomedical materials. The double extruded JDBM-2 alloy with excellent biocompatibility, suitable
mechanical properties (yield strength: 2766MPa, elongation: 34.33.4%) and service life-time (up to
6-month) represents a major breakthrough to conventional stent materials and a significant advance towards
the goal of successful clinical translation of full-degradable Mg-based cardiovascular stent.

These findings are consistent with those of in vitro studies that the JDBM-2 is well compatible with endothelial
cells and causes minor inflammatory response, which represents the demonstration of excellent tissue response
to this stent material.

Discussion
A stent is a small mesh-like tubular scaffold which is mounted on a balloon and expanded inside the target
coronary artery to keep the lumen open. Medical metals and polymers are the most common materials used in
manufacturing cardiovascular stent over the past decades5,7,9,10,1215. Figure9 illustrates satisfactory stent material
(shadow area) required for clinical application and limitations of current biomedical materials. The candidate
materials for stent application should meet at least the three following requirements: (1) biocompatibility (cellular
activity >80%): the material and its products do not pose a significant risk of toxicity to cells and must be well
tolerated by the patient; (2) mechanical properties (yield strength >200 MPa, elongation >20%): the material
should have sufficient strength for load-bearing application and also have acceptable ductility for a compliance
match between stented and non-stented vessel areas; (3) service lifetime (up to 612 months): the material should
stay in the diseased vessel limited to a period of 612 months and then can be absorbed or excreted by the
surrounding tissues and the metabolic system after fulfilling its functionality. The application of biodegradable
polymers and irons is limited by the relatively low mechanical strength and long corrosion period (service life-
time), respectively. While traditional stent material such as 316L stainless steel is challenged by the long-term
endothelial dysfunction, chronic inflammation and delayed re-endothelialization, especially inability to adapt to
growth in young patients. In this case, Mg alloys appear to be promising candidates that can potentially address
the above challenges as temporary structural biomaterial for cardiovascular stent application due to the intriguing

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combination properties of biocompatibility, biodegradability and mechanical performance1921,23,24,26. In the pres-

ent study, we carried out systematic investigation with the purpose of identifying whether the double extruded
JDBM-2 Mg alloy is an alternative biomaterial which is suitable for real clinical biodegradable stent application.
Stent materials with high strength and ductility allow the intraoperative mechanical deformation and a suffi-
cient radial support to overcome the problem of elastic recoil following balloon expansion. High-strength mate-
rials also enable the design of thinner stent struts, which is beneficial for more flexible devices and lower vascular
trauma9,32,33. As a new and effective processing technology, double extrusion was employed in this study to
improve the mechanical properties of Mg alloys. The UTS, YS and elongation of solution treated (T4) JDBM-2 are
increased by 306%, 70 and 209% respectively after double extrusion. Grain refinement is an effective way to
strengthen mechanical properties of Mg alloys. However, long elongated grains were observed in the once
extruded condition (Fig.1b). In the basal slip system, these grains aligning along <1120> crystal direction with
their c-axis perpendicular to the extrusion direction are favorably oriented to accommodate extrusion strains.
While undergoing large deformation without twinning and latent hardening, once extruded Mg alloys do not
have large enough stored plastic energy to trigger recrystallization, thus long elongated grains are easily formed
in the microstructures of these alloys34,35. After double extrusion, the long elongated grains disappear due to the
dynamic recrystallization triggered by the stored plastic energy generated in the process of hot extrusion, and the
matrix grains are finer and more homogeneous, which is beneficial for the improvement of ductility. The refined
homogeneous equiaxed grains achieved in complete dynamic recrystallization and the precipitation of nano par-
ticles (precipitated phases) in grain interiors and boundaries (Fig.1d) lead to significant increase in yield strength.
There are many publications on the investigation of mechanical properties of Mg alloys at room temperature.
Researchers have reported remarkably high tensile yield strengths (e.g. yield strength >350MPa) of Mg alloys
while the elongation is relative low (e.g. elongation <10%)3638. Few of them can satisfy both the high yield
strength (>270MPa) and elongation (>30%) simultaneously. In our study, the room temperature mechanical
properties of the JDBM-2 alloy are fascinating. The yield strength and elongation of the JDBM-2 after double
extrusion are 2766MPa and 34.33.4% respectively, which is close to those of clinical used medical stainless
steel 316L39.
Corrosion is normally a drawback in engineering, however, the corrodibility of Mg alloy is of considera-
ble interest for their application as degradable implants. Unfortunately, localized corrosion of conventional Mg
alloys such as AZ31 (Fig.2a) may lead to undesirable phenomenon such as losing efficacy or even fracture after
implantation. The corrosion mechanism of Mg alloys is profoundly influenced by the second phases/precipitated
phases and their distribution4042. Mg alloys with a highly stable interface with the physiological environment
would slow down the tendency of localized corrosion through reducing electron exchange between the metal
and the adsorbed biological medium43. After double extrusion, a dense and fine dispersion of second phases/
precipitated phases distributes in the JDBM-2 alloy (Fig.1d). The slight potential difference (~25mV) between
Mg12Nd and the -Mg matrix is anticipated to lead to increase in the tendency of interface stability in the physio-
logical environment and consequent improvement in homogeneous degradation of the alloy44. Hence, we further
conducted the cyclic polarization measurement to research the propensity of the double extruded JDBM-2Mg
alloy to undergo local corrosion in artificial plasma (Fig.2e). Generally, in a cyclic polarization curve, the forward
scan represents the polarization behavior of the non-corroded areas while the reverse scan is associated with the
performance of the corroded areas. Due to the galvanic corrosion effect, an area with a more negative potential
that acts as anode is corroded and an area with a more positive potential that acts as cathode is protected. In our
study, the corrosion potential of the reverse scan in the JDBM-2 alloy is more positive than that on the forward
scan (E+ < E), which means the corrosion of the corroded area on JDBM-2 alloy is likely to be suppressed by
the non-corroded area, while the non-corroded area is prone to be eroded due to a lower corrosion potential.
This result can lead to reduced propensity of localized corrosion attack in the JDBM-2 alloy due to the anode and
cathode switch in the galvanic corrosion process. Thus, the corrosion morphology observation (Fig.2b) and the
cyclic polarization characterization (Fig.2e) confirm the fact that the JDBM-2 exhibits homogeneous corrosion
in artificial plasma, which is desirable for biodegradable medical devices to avoid local stress concentration and
enables researchers to predict the accurate degradation period in vitro and in vivo. Based on the calculation of
corrosion rate, the lifetime of the JDBM-2 stent (~0.2mm thickness) is estimated up to 6-month, which is suitable
for blood vessel remodeling. Therefore, the improvement in interface stability of JDBM-2 in the physiological
environment results in enhanced homogeneous corrosion tendency and potentially long-term durability in vivo,
which can be further confirmed by the implantation of stent in animal model (Fig.7).
The evaluation of biological responses to implants is a measurement of the magnitude and duration of the
adverse alterations in homeostatic mechanisms that determine whether the medical device can be completely
compatible with the surrounding tissues without causing significant damage to the vital organs. From clinical
and regulatory perspective, the corrosion kinetics of Mg and its alloys exceeds that of toxic tolerance, resulting
in potentially harmful perturbations to the physiological environment and a threat to neighboring tissues and
organs. Mg alloy with a stable interface with the physiological environment would slow down the corrosion
process, reduce the accumulation of ions and thus, improve the biocompatibility and bio-efficacy of the implant.
As mentioned above, JDBM-2 with a stable interface in the physiological environment achieved through fine
dispersion of second phases/precipitated phases with slight potential difference compared to the -Mg matrix
results in homogeneous degradation mechanism and slow corrosion rate, which is beneficial for the improvement
of biocompatible outcome. For stent application, Mg alloy with a controlled degradation rate appears to have a
positive effect on HUVECs deposition, proliferation and growth and potentially aids in re-endothelialization of
the implant into the denuded artery in vivo. Enhanced endothelial cell adhesion and rapid re-endothelialization
process has been suggested as a method for increasing efficacy of vascular stents through preventing thrombosis
and reducing FBR45. At last, we performed in vivo assessment via implantation of JDBM-2 stent to the animal

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model. The result confirms excellent tissue compatibility (Figs68) and up to 6-month structural and mechan-
ical integrity of the stent in vivo (Figs6,7), suggesting significant advances have been made towards the goal of
successful clinical translation of biodegradable Mg-based cardiovascular stent. In summary, the double extruded
JDBM-2 alloy with excellent biocompatibility, suitable mechanical properties and service life-time (Fig.9) rep-
resents a major breakthrough to conventional stent materials and shows great potentiality for large-scale clinical
application.

Conclusions
Although Mg alloys have been investigated as biomaterials for over a decade, to the best of our knowledge, this is
the first report to show that our Mg alloy Mg-2.2Nd-0.1Zn-0.4Zr (JDBM-2) with excellent cellular compatibility
can integrate the mechanical properties of medical stainless steel and the degradation durability of polymer for
temporary cardiovascular stent application. The yield strength and elongation of the JDBM-2 are 2766MPa
and 34.33.4% respectively. A stable interface with the physiological environment of the JDBM-2 leads to sig-
nificant improvement in homogeneous degradation mechanism and reduced corrosion rate. In vitro cytotoxicity
test shows the JDBM-2 has negligible adverse effect on HUVECs viability and growth. This alloy also exhibits
compromised FBR in comparison with 316L stainless steel via investigation of human peripheral blood derived
macrophage adhesion, FBGC formation and inflammatory protein production. The implantation of the JDBM-2
stent confirms excellent tissue compatibility and up to 6-month structural and mechanical integrity in vivo, repre-
senting a significant step towards the goal of successful clinical translation of Mg alloys in biodegradable medical
devices, and showing great potential to be alternative to conventional stent materials.

Materials and Experiments

Materials and heat treatments. Mg-2.25Nd-0.11Zn-0.43Zr (wt.%, JDBM-2) was cast by semi-continuous
casting with high purity Mg (99.99%), Zn (99.995%), Mg-25%Nd (impurities0.1%) and Mg-30%Zr (impu-
rities0.5%). The cast ingot was solution (T4) treated at 540C for 10h in a protected atmosphere, quenched into
water at room temperature. The ingot in T4 condition was first extruded at 290C with an extrusion ratio of 8 and
extrusion speed of 50mm/s for the first extrusion, followed by the second extrusion at 320C with an extrusion
ratio of 9 and extrusion speed of 18mm/s.

Microstructures and Mechanical properties. Specimens (125mm) were grounded with SiC paper,
mechanically polished using 3.5 and 0.5m diamond paste and light MgO solution, and then were etched with
acid solution (10ml acetic acid, 4.2g picric acid, 70ml ethanol, and 10ml distilled water). Tensile test speci-
mens with the dimensions of 3.5mm width, 2mm thickness, and 30mm gauge length were cut parallel to the
extrusion direction, polished with SiC paper and 3.5m diamond paste, respectively. The microstructures were
observed using optical microscope (Zeiss, Oberkochen, Germany) and field emission scanning electron micro-
scope (FE-SEM, SIRION 200, FEI, America), coupled with energy-dispersive X-ray spectrometry (EDX, INCA,
Oxford, U.K). Tensile tests were carried out at room temperature on a uniaxial tensile testing machine Zwick
Z100, coupled with a contact extensometer, with primary strain rate of 1mm/min. The stress-strain curve was
obtained under a uniaxial loading.

In vitro degradation tests. In vitro degradation tests were carried out in artificial plasma (composi-
tion: 6.8g/L NaCl, 0.4g/L KCl, 2.2g/L NaHCO3, 0.1g/L MgSO4, 0.2g/L CaCl2, 0.126g/L Na2HPO4, 0.026g/L
NaH2PO4 and 1.0g/L glucose) at 370.5C for 120h. The volume of artificial plasma was calculated based on
a volume-to-sample area ratio of 40mL/cm2. After immersion, the specimens were cleaned using a standard
chromium trioxide (Cr2O3) solution recommended in ASTM G1-90 to remove the corrosion products. Four
specimens for each group were tested, and the corrosion rate (CR) determined by mass loss was calculated using
the following equation:

8.76 104 w
CR =
At
where, w (g) is the weight change, A( cm2) is the surface area of the specimen, t (h) is the immersion time and
(gcm3) is the density of the alloy. Cyclic polarization measurement of Mg alloy was carried out in artificial
plasma by Advanced Electrochemical System (PARSTAT 2273, Princeton) with a polarization scan at a rate of
1mV/s.

In vitro cytotoxicity evaluation. Mg alloy extract was prepared using EBM (Lonza, Cat. # CC-3156)
serum free medium with the surface area of extract medium ratio of 1.25cm2/mL. 100L cell suspension with the
density of 2 104/mL was cultivated in 96-well cell culture plates in each well. After incubation for 24h to allow
attachment, the EBM medium was replaced with 100L of extracts with the dilution of 10 and 50% concentra-
tions. After incubated for 1, 3, 5 days, cytotoxicity evaluation was determined using a standard MTT method.
The O.D. (optical density) intensity of supernatant was measured at a wavelength of 490nm by iMark Microplate
Reader (BioTek instruments). Human endothelial cells incubated for a period of 24h were stained with Pholloidin
(Life Technologies-Invitrogen, Cat. # A22282) and DRAQ5 (Cell Signaling Technology, Cat. # 4084S) to identify
cell morphologies. Proliferating endothelial cells were monitored by 5-Bromo- 20-deoxy-uridine (BrdU) incor-
poration into nuclei of dividing cells for 30min, followed by BrdU Mouse mAb Labelling and Anti-Mouse IgG
(H+L) Detection (Cell Signaling Technology, Cat. # 6813).

In vitro macrophage assay and protein secretomic analysis. To investigate the expression of
cytokine and chemokine from macrophages/FBGCs during FBR in vitro, human macrophages were cultured

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www.nature.com/scientificreports/

on the material surface based on a metal-based cell culture system (Scheme 1). Human monocytes were iso-
lated from peripheral blood and differentiated into macrophages in the presence of granulocyte macrophage
colony-stimulating factor (GM-CSF, Biosystems, Cat. # GMF-H4214). Macrophages adhesion and fusion were
investigated by culturing cells directly on substrates with cell density of 2 105/mL. The adherent cells were
double-stained with phalloidin (Life Technologies-Invitrogen, Cat. # A22282) and DAPI (Invitrogen, cat. #
D3571). Cytokine and chemokine production of macrophages/FBGCs in response to JDBM-2 and stainless steel
substrates was determined by enzyme linked immunosorbent assay (ELISA).

Stent implantation. Animal experiments were conducted under the NIH Guide for Care and Use of
Laboratory Animals and approved by the Animal Ethics Committee of Zhongshan Hospital. 34 months old, sex
unlimited, healthy and clean New Zealand white rabbits weighing 2.53.5kg were brought from the laboratory
animal center of Zhongshan Hosptial, Fudan University, China. JDBM-2 stent with an original dimension of
214mm was fabricated in Shanghai Jiao Tong University (SJTU), China. Commercial 316L stainless steel
stent was studied as control group. Serial angiography and the follow-up intravascular ultrasound (IVUS) were
performed to examine the safety and efficiency of the stents. Histologic examination of stents via HE staining and
SEM were used to determine the tissue response to the implant and the re-endothelialization process.

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Acknowledgements
This work was supported by the Yale University new faculty startup fund (PI: R.F.). Research at Shanghai Jiao
Tong University (SJTU) was supported by the National Key Technology R&D Program of the Ministry of Science
and Technology (2012BAI18B01). This project was also supported by the National Basic Research Program
(973 Program) of China (2011CB503905), the National Natural Science Foundation of China (81370323) and
China Postdoctoral Science Foundation funded project (2016M590368).

Author Contributions
J.-B.G. and W.-J.D. conceived the whole project. G.-Y.Y. and C.-L.S. designed experiments for materials synthesis,
heat treatment, mechanical property measurements and degradation characterizations. L.M., X.-B.Z., L.Z. and
J.P. performed mechanical performance tests and corrosion behavior evaluations. L.M. and R. F. designed the
in vitro cell assays. L.M., M.K. and Y.W performed in vitro cell assay experiments and analyzed data. L.S. and
J.-H.C. performed the animal experiments, the relevant data collection and data analysis. All the authors
contributed to the writing of manuscript. L.M. and L.S. contributed equally to this work.

Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing Interests: The authors declare no competing financial interests.
How to cite this article: Mao, L. et al. A promising biodegradable magnesium alloy suitable for clinical vascular
stent application. Sci. Rep. 7, 46343; doi: 10.1038/srep46343 (2017).
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Scientific Reports | 7:46343 | DOI: 10.1038/srep46343 12