Acta Biomaterialia 71 (2018) 200–214

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Full length article

In vitro and in vivo studies on zinc-hydroxyapatite composites as novel

biodegradable metal matrix composite for orthopedic applications
Hongtao Yang a,1, Xinhua Qu b,1, Wenjiao Lin c, Cong Wang d, Donghui Zhu e, Kerong Dai b,⇑,
Yufeng Zheng a,f,⇑
a
Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China
b
Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine,
Shanghai 200011, China
c
R&D Center, Lifetech Scientific (Shenzhen) Co Ltd, Shenzhen 518057, China
d
Department of Interventional Radiology and Vascular Surgery, Peking University Third Hospital, Beijing 100191, China
e
Department of Biomedical Engineering, College of Engineering, University of North Texas, Denton, TX 76207, USA
f
International Research Organization for Advanced Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-Ku, Kumamoto 860-8555, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Recent studies indicate that there is a great demand to optimize pure Zn with tunable degradation rates
Received 17 December 2017 and more desirable biocompatibility as orthopedic implants. Metal matrix composite (MMC) can be a
Received in revised form 6 February 2018 promising approach for this purpose. In this study, MMC with pure Zn as a matrix and hydroxyapatite
Accepted 3 March 2018
(HA) as reinforcements were prepared by spark plasma sintering (SPS). Feasibility of novel Zn-HA com-
Available online 9 March 2018
posites to be used as orthopedic implant applications was systematically evaluated. After sintering, HA
distributed in the Zn particle boundaries uniformly. Corrosion tests indicated that the degradation rates
Keywords:
of Zn-HA composites were adjustable due to the biphasic effects of HA. Zn-HA composites showed signif-
Zinc
Hydroxyapatite
icantly improved cell viability of osteoblastic MC3T3-E1 cells compared with pure Zn. Both pure Zn and
Composite composites exhibited a low thrombosis risk and hemolysis rates while a Zn ion concentration-dependent
Degradation effect was found on coagulation time. An effective antibacterial property was observed as well. The vol-
Biocompatibility ume loss of pure Zn and Zn-5HA composite was 1.7% and 3.2% after 8 weeks’ implantation. Histological
analysis found newly formed bone surrounding pure Zn and Zn-5HA composite at week 4 and increased
bone mass over time. With prolonged implantation time, Zn-5HA composite was more effective on stim-
ulating new bone formation than pure Zn. In summary, MMC is a feasible way to design Zn based mate-
rials with adjustable degradation rates and improved biocompatibility.

Statement of Significance

Biodegradable zinc materials are promising candidates for the new generation of orthopedic implants.
However, the slow degradation rates and unsatisfactory cytocompatibility of pure Zn in bone environ-
ments limit its future clinical applications. Generally, alloying is a common way to improve the perfor-
mance of pure Zn. In this study, metal matrix composite was chosen as a novel strategy to solve the
problems. Hydroxyapatite, as a bioactive component, was added into Zn matrix via spark plasma sinter-
ing. We find that Zn-HA composites exhibited adjustable degradation rates and improved biocompatibil-
ity both in vitro and in vivo. This study provides exhaustive and significant information including
microstructure, mechanical performance, degradation behavior, biocompatibility, hemocompatibility
and antibacterial property for the future Zn based implants design.
Ó 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

⇑ Corresponding authors at: Department of Materials Science and Engineering,
College of Engineering, Peking University, Beijing 100871, China (Y. Zheng).
E-mail addresses: krdai@163.com (K. Dai), yfzheng@pku.edu.cn (Y. Zheng).
1
These two authors contributed equally to this study.
Biodegradable metals have been viewed as ideal materials for
applications in orthopedics due to their desirable biodegradability,
excellent mechanical property and good biocompatibility. Zinc, as

https://doi.org/10.1016/j.actbio.2018.03.007
1742-7061/Ó 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214
a novel candidate, has attracted increasing interests recently.
Unlike magnesium, the degradation of zinc does not create hydro-
gen gas pockets caused by fast corrosion which may interfere with
the bone healing process, resulting in callus formation and cortical
defects [1]. Also, unlike iron, zinc exhibits a better in vivo degrada-
tion behavior without leaving voluminous corrosion products that
are hard to be eliminated by human body [2]. Apart from the favor-
ite degradation behavior, the importance of zinc in human nutri-
tion has also been proved. Zinc, as an essential element in the
human body, participates in various basic biological functions
including nucleic acid metabolism, signal transduction, apoptosis
regulation and gene expression in addition to interacting with a
lot of organic ligands [3]. More importantly, zinc plays an impor-
tant role in the growth and mineralization of bone tissue. Zinc
stimulates bone formation and mineralization; the metal directly
activates aminoacyl-tRNA synthetase in osteoblastic cells and
stimulates cellular protein synthesis. In addition, zinc inhibits
osteoclastic bone resorption by inhibiting osteoclast-like cell for-
mation from marrow cells [4]. Zinc also plays a role in the preser-
vation of the bone mass. However, the reported degradation rates
of pure Zn in bone environments are relatively slow. The in vitro
and in vivo degradation rates of pure Zn were approximately
0.06–0.08 mm/year and 0.2 mm/year [5,6], respectively. For bone
fracture fixation, the mechanical support of biodegradable
implants should sustain for 12–24 weeks depending on the clinical
conditions [7]. Thereafter, while the mechanical integrity
decreases, the degradation should progress at a sufficient rate
without causing an intolerable host response. Considering a typical
screw 5 mm in diameter and 50 mm in length for the fixation of a
fracture bone. A simple calculation reveals that the complete
degradation of this screw takes more than a decade. In addition,
the type of environmental tissue plays an important role in the
degradation process of biodegradable implants. Soft tissue may
cause more rapid degradation due to the superior vascularization
or the avoidance of changes in local pH value [8,9]. For example,
a faster degradation of intramedullary areas compared to cortical
areas was observed in Mg alloys, which attributed to the enhanced
vascularization of the bone marrow [1]. Therefore, it is necessary to
develop Zn based materials with adjustable degradation rates. Pure
Zn also showed significant toxicity to normal human osteoblast
cells. Cell viability dropped to lower than 50% after 7 days’ culture
with pure Zn extracts. Cells showed a decrease in both the size and
the actin microfilament intensity after exposing to Zn. Moreover,
significant DNA damage was detected in cells after 7 days incuba-
tion with extracts [10]. Pure Zn exhibited toxicity in human
endothelium-derived cells and L292 cells as well [11,12].
Metal matrix composite (MMC) can be a feasible approach to
improve the performance of pure Zn. The advantages of MMC are
adjustable corrosion properties as well as biocompatibility by
choosing the appropriate composites. The interaction of MMC with
surrounding tissues can be optimized by varying the constituents,
the type and distribution of the reinforcing phase. Hydroxyapatite
(HA) is a well-known bioceramic with bioactivity that supports cell
proliferation, bone ingrowth and osseointegration [13,14]. HA has
similar chemical and crystallographic structures to bone, which
can form a chemical bond with osseous tissue and act like nucle-
ations for new bone [15]. Its main components, Ca and P, are both
essential elements that maintain physiological homeostasis pro-
cesses [16]. Gu et al. [17] prepared Mg/HA composite and found
that HA created more anodic sites in Mg matrix forming galvanic
coupling which accelerated the corrosion rate of Mg. Witte et al.
[18] fabricated the AZ91D/HA metal matrix composite by powder
metallurgy method and found that the composite was cytocompat-
ible with better corrosion behavior compared with AZ91D alloy.
Addition of bioceramics (HA, TCP, BCP) into iron resulted in
increased corrosion rates and improved cellular activity compared
201
to pure iron [19]. Therefore, improvements in degradation and bio-
compatibility can be expected by incorporating HA into pure Zn.
In the present study, Zn-HA composites was fabricated by spark
plasma sintering (SPS). The microstructure, mechanical properties,
in vitro corrosion behavior, cytotoxicity, hemocompatibility,
antibacterial property and in vivo test have been systematically
investigated to evaluate the performance of Zn-HA composites as
biodegradable implants for orthopedic applications.
2. Materials and methods
2.1. Material preparation
Pure Zn powder (99.9%,
140+325 mesh, Alfa) and hydroxyap-
atite (HA) powder (99%, 150 nm in length, Beijing Daoking) were
used as raw materials. Pure Zn powders were mixed with HA pow-
ders (1, 5 and 10 wt.%) in planet grinding machine (XM-4, KELI
CERAMICS) under argon atmosphere and ball milling for 1 h at
300 rpm. Then, pure Zn powders, the mixed Zn-HA powders were
put into a 30 mm diameter graphite die and sintered under vac-
uum by SPS-1050 (Sumitomo Coal Mining Company, Ltd.) with sin-
tering temperature of 380 °C, sintering pressure of 40 MPa, and
holding time of 6 min. Pure Zn prepared by SPS served as control.
The as-sintered pure Zn and Zn-HA composites were cut into disk
samples (U10  1 mm) and cylinders (U2  4 mm and U2  5
mm), respectively. Disk samples were used for density measure-
ment, microstructure characterization, microhardness, electro-
chemical tests, immersion tests, biocompatibility,
hemocompatibility and antibacterial tests. Cylinders were used
for compressive (U2  4 mm) and animal tests (U2  5 mm). Each
sample was mechanically polished to 2000 grit, then ultrasonically
cleaned in acetone and ethanol, then dried in the open air. Before
cytotoxicity, antibacterial and animal tests, samples were sterilized
with ultraviolet radiation for at least 4 hours each side.
2.2. Microstructure characterization
An electronic balance (XS105, METTLER TOLEDO) attached with
density accessories was used to measure the density of all samples.
Samples were further polished by 0.1 lm diamond paste and
cleaned in acetone. After samples were etched with a 4% HNO3/
alcohol solution, an optical microscope (Olympus BX51 M) was
used to observe the metallographic structure. X-ray diffractometer
(XRD, Rigaku DMAX 2400, Japan) using CuKa radiation was
adopted to identify the constituent phases of pure Zn and Zn-HA
composites with scanning range from 20° to 80° and scan rate of
6° min
1. Energy dispersive spectrometer (EDS, Bruker QUANTAX,
Germany) was used for the analysis of chemical compositions.
2.3. Mechanical tests
Microhardness and compressive tests were conducted to evalu-
ate the mechanical properties of pure Zn and Zn-HA composites.
Microhardness test was carried out by a microhardness tester
(SHIMADZUHMV-2t) measuring Vickers hardness with loading
force of 0.1 kN and dwell time of 15 s. Instron 5969 universal test
machine was used for the compressive test. The specimens were in
the form of cylinder 2 mm in diameter and 4 mm in length. Com-
pression strain rate was 2  10
4 s
1. An average of at least three
measurements was taken for each group.
2.4. Electrochemical measurements
The electrochemical tests were conducted with an electrochem-
ical workstation (Autolab, Metrohm, Switzerland) at room temper-
202 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214
ature in Hank’s solution (NaCl 8.00 g L
1, KCl 0.40 g L
1, CaCl2 0.14
g L
1, NaHCO3 0.35 g L
1, glucose 1.00 g L
1, MgCl26H2O 0.10 g
L
1, MgSO47H2O 0.06 g L
1, Na2HPO412H2O 0.06 g L
1 and KH2-
PO4 0.06 g L
1, pH 7.4). A three-electrode cell with a platinum
counter electrode and a saturated calomel electrode (SCE) as the
reference electrode was utilized for electrochemical tests. The
open-circuit potential (OCP) of each sample was monitored for
5400 s. Electrochemical impedance spectroscopy (EIS) measure-
ment was carried out by applying 10 mV perturbation with the
measuring frequency ranging from 105 Hz down to 10
2 Hz. After-
wards, potentiodynamic polarization test was carried out at a scan-
ning rate of 1 mV s
1. The measurement area was 0.2826 cm2 (U 6
mm). At least five measurements were taken for each sample
group. Corrosion parameters including open-circuit potential
(OCP), corrosion potential (Ecorr) and corrosion current density
(icorr) were analyzed by linear fit and Tafel extrapolation to the
cathodic and anodic parts of polarization curves.
2.5. Immersion tests
Immersion tests were carried out in Hank’s solution at 37 °C for
50 days. The pH value of the solution was recorded by a pH meter
(Mettler FiveEasy pH FE20K) during immersion. After 50 day’s
immersion, samples were removed from Hank’s solution, gently
rinsed with distilled water and dried in air. The corroded surface
morphology was characterized by scanning electron microscopy
(SEM, Hitachi S-4800, Japan) and optical microscope (Olympus
BX51 M). X-ray diffractometer (XRD, Rigaku DMAX 2400, Japan)
using CuKa radiation was adopted to identify the constituent
phases of corrosion products. Samples were embedded in epoxy
and the cross sections were polished for SEM observation and
EDS analysis. The weight loss of the samples was measured on
an electronic balance (XS105, METTLER TOLEDO) with a measuring
sensitivity of 0.1 mg after removal of corrosion products. An aver-
age of five measurements were taken for each group. The in vitro
corrosion rate was calculated according to the equation: C = Dm/
qAt, where C is the corrosion rate in mm year
1, Dm is the weight
loss, q is the density of the material, A is the initial implant surface
area and t is the implantation time. Related ion concentrations in
solution were measured by Inductively Coupled Plasma Optical
Emission Spectroscope (ICP-OES, iCAP6300, Thermo).
2.6. In vitro tests
2.6.1. Cell viability
MC3T3-E1 cells were cultured in Alpha-minimum essential
medium (MEM), 10% fetal bovine serum (FBS), 100 U mL
1 peni-
cillin and 100 lg mL
1 streptomycin at 37 °C in a humidified atmo-
sphere of 5% CO2 (Alpha-minimum essential medium (MEM), fetal
bovine serum (FBS), penicillin and streptomycin were purchased
from Gibco BRL (Gaithersburg, MD, USA)). The cytotoxicity test
was evaluated using the 100% extracts of metal plates, prepared
with a surface area to extraction medium ratio of 1.25 mL cm
2
in a humidified atmosphere, with 5% CO2 at 37 °C for 24 h. The
supernatant fluid was withdrawn, centrifuged and kept at 4 °C
prior to use. Cells were incubated in 96-well culture plates at a
density of 3  104 cells mL
1 in each well and incubated for 24 h
to allow attachment. The medium was then replaced with 100 lL
sample extracts. The culture medium was used as the negative
control and the culture medium added with 10% dimethylsulfoxide
(DMSO, Invitrogen, USA) as the positive control. After 1, 2 and 4
days of incubation, 10 lL Cell Counting Kit-8 (CCK-8, Dojindo,
Kumamoto, Japan) was added to each well and incubated for 1 h.
The spectrophotometrical absorbance of samples was measured
at 450 nm using a microplate reader (Bio-RAD680).
2.6.2. Hemolysis and platelet adhesion
Healthy human blood (anticoagulant with 3.8% citric acid
sodium) extracted from volunteers was diluted by physiological
saline with a volume ratio 4:5. Pure Zn and Zn-HA composites were
installed in centrifugal tubes with 10 mL physiological saline and
water bathed at 37 °C for 30 min. Then, 0.2 mL diluted blood was
added to each tube and incubated at 37 °C for 60 min, 10 mL deion-
ized water with 0.2 mL diluted blood as the positive control and 10
mL physiological saline with 0.2 mL diluted blood as the negative
control. Afterwards, samples were removed and tubes ware cen-
trifuged at 3000 rpm for 5 min. Supernatant was transferred to
96-well plates, the absorbance (OD) was determined by a micro-
plate reader (Bio-RAD680) at a wavelength of 545 nm. Hemolysis
of samples was calculated by the formula:
ODðtestÞ
ODðnegative controlÞ
Hemolysis ¼  100%
ODðpositive controlÞ
ODðnegative controlÞ
For platelet adhesion, whole blood from healthy human was
centrifuged at 1000 rpm for 10 min. Platelet rich plasma (PRP)
was obtained from the upper fluid. Samples were moved into 24-
well plates and 0.2 mL PRP was added to each well, incubated at
37 °C for 1 h. After gently flushed by phosphate buffered saline
(PBS), platelets on samples were fixed with 2.5% glutaraldehyde
solution at room temperature for 2 h, then dehydrated with gradi-
ent alcohol solution (50%, 60%, 70%, 80%, 90%, 95% and 100%) for 10
min, and finally dried in desiccator for 2 days. The morphology of
platelet adhered on the specimens was coated by gold and
observed by SEM (Hitachi S-4800, Japan).
2.6.3. Coagulation time
Platelet poor plasma (PPP) was prepared by centrifuging the
citrated whole blood at 3000 rpm for 15 min and then the super-
natant was collected. The plasma was added into samples with a
ratio 500 lL cm
2 and water bathed at 37 °C for 30 min. Then the
assays including activated partial thromboplastin time (APTT), pro-
thrombin time (PT) and thrombin time (TT) were measured at the
Third Hospital of Peking University with corresponding reagents
added. Zn ion concentrations in PPP were measured by ICP-OES.
2.6.4. Antibacterial test
S. aureus (ATCC 25922) was used as a model bacterium to deter-
mine the antibacterial properties of pure Zn and Zn-HA composites
with Ti6Al4V as control. S. aureus was cultured in a 37 °C shaker
incubator at 220 rpm for 16 h in 20 mL of the Luria-Bertani (LB)
medium containing 10 g L
1 peptone, 10 g L
1 NaCl and 5 g L
1
beef extract. The density of the bacteria cultured in the medium
was quantified by absorption spectroscopy at a wavelength of
about 600 nm (OD600) after performing colony counts on different
concentration bacterial suspensions. OD600 was converted to col-
ony forming units (CFUs) as follows: OD600 = 0.1 contained 108
CFUs. Serial dilution of the bacteria was performed in the LB med-
ium to achieve a final bacterial cell suspension of 1  106 and
1  107 CFUs per mL, respectively. Then, 1 mL of the bacterial cell
suspension at a concentration of 107 CFU per ml was added to each
sample in 24-well plates. The plates were incubated at 37 °C in a
humidified atmosphere with 5% CO2 for 24 h. After the incubation
procedure, 100 lL bacterial suspension were plated in duplicate on
the agar plates. CFUs were determined on the plates after incuba-
tion for another 24 h. Meanwhile, samples were gently rinsed three
times with PBS in order to eliminate the non-adherent bacteria and
then transferred to new tubes containing 5 mL of PBS. The samples
in the tubes were then sonicated for 5 min in a 40 W ultrasonic
bath at a nominal frequency of 43,000 Hz followed by vortexing
for 30 seconds to remove the adherent bacteria into the PBS solu-
tions. The viable organisms in the PBS buffers were quantified by
 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214
plating serial dilutions on the LB agar plates. The plates were incu-
bated for 24 h at 37 °C in a humidified atmosphere with 5% CO2,
and the colony-forming units were counted with the naked eye.
The antibacterial rates for planktonic bacteria and adhered bacteria
were calculated based on the formulas: (1) Antibacterial rate for
planktonic bacteria (Rp) (%) = (B
A)/B  100% and (2) Antibacte-
rial rate for adherent bacteria (Ra) (%) = (D
C)/D  100%. Here,
A indicates the average number of viable bacteria in the culture
medium incubated with pure Zn and Zn-HA composites, B is the
average number of viable bacteria in the culture medium incu-
bated with Ti6Al4V, C is the average number of viable bacteria
adhered on pure Zn and Zn-HA composites, D is the average num-
ber of viable bacteria adhered on Ti6Al4V.
2.7. In vivo tests
2.7.1. Surgical implantation
All the animal procedures and experiments were approved by
the Animal Ethical Committee at the Ninth People’s Hospital affil-
iated to Shanghai Jiaotong University, School of Medicine (Shang-
hai, China). A rat femur condyle model was used and the surgical
procedures were conducted under sterile conditions. The rats were
anesthetized by intraperitoneal injection of ketamine (10 mg/kg,
Shanghai Ziyuan Pharmaceutical Co. Ltd, Shanghai, China) and 2%
xylazine (5 mg/kg, BayerAG, Leverkusen, North Rhine-Westphalia,
Germany). Buprenorphine (Temgesic, Reckitt & Cloman, Hull, UK)
was given subcutaneously by a dose of 0.3 mg/kg for postoperative
analgesia. Each rat was immobilized in maximally flexed position
with the knee joint, while both legs were shaved and depilated.
Afterwards, a 10 mm longitudinal incision was made along the lat-
eral side of the extensor mechanism around the knee joint. The
femoral lateral condyle in each rat was sufficiently exposed with
the knee in flexion, and a hole (2 mm in diameter and 5 mm in
depth) was drilled through the distal of the epiphysis gap in the
bone. After washing the bone cavity with normal saline to remove
debris, pure Zn or Zn-5HA composite were inserted into the left or
right femur condyle of rats in a blind manner. A total of 32
implants were used. Two time points were selected (week 4 and
8) and 8 implants were implanted at each time point in each group.
Each rat received one implant. The wound was closed carefully.
Thirty-two male Sprague Dawley rats aged 3 months and weighed
by an average of 200 g (180–235 g) were randomized to either
pure Zn or Zn-5HA group. Just after the surgery, an X-ray scan
was performed to control the position of the implant. After surgery,
the rats were housed in groups of four in clear plastic cages on
standard bedding in ventilated rooms with a 12 h day/night cycle
and an ambient temperature of 21 °C, and given access to water
and standard laboratory pellets. At 4 or 8 weeks, the rats were sac-
rificed and the rat condyles were explanted and fixed in 10% neu-
tral formalin buffer for micro-CT analysis and histomorphometric
observation.
2.7.2. X-ray scanning
The X-ray machine (Digital Diagnost, Philips, Amsterdam,
Netherlands) at the operation conditions was set as follows: 52
kV and 3.2 mAs. The X-ray scanning was then performed at week
0 (n = 32), week 4 (n = 32) and week 8 (n = 16) to evaluate the
implants.
2.7.3. Micro-CT analysis
Micro-CT analysis of pure Zn and Zn-HA composite implants
was made using Computer Tomographic data obtained with a Sky-
scan 1172 micro-CT system (Bruker micro-CT N.V., Kontich, Bel-
gium). A total of 8 implants were scanned at each time point in
each group. Explanted rat condyles were examined with a 20 lm
resolution protocol (100 kV, Al + Cu filter, 0.4 degree rotation step,
203
frame averaging of 2 degree, 360 degree rotation) at week 4 and 8,
respectively. CT images were reconstructed using Skyscan NRecon
software (Bruker micro-CT N.V., Kontich, Belgium). The volumes of
remained metallic implants were determined in CTAn software
and their 3D images were yield by CTVol and CTVox software (Bru-
ker micro-CT N.V., Kontich, Belgium).
2.7.4. Histomorphometric analysis
The retrieved implants were processed for hard tissue (n = 16)
and soft tissue (n = 16) staining. After fixation, the implants were
rinsed in water, dehydrated in ethanol, cleared in xylene, and
embedded in methyl methacrylate. Each implant was cut along
its vertical axis giving 5–6 sections per implant. The three most
central sections from each specimen, which correspond to full-
length sections, were kept for grinding to 100 lm thickness, pol-
ishing, staining and histological analysis. Selected sections were
ground to a thickness of 100 lm, polished and stained with van
Gieson Picrofuschin. At least 8 sections were selected for hard tis-
sue histological analysis at each time point in each group. For soft
tissue staining, the samples were decalcified in 10% EDTA for 6
weeks, and then embedded in paraffin. Histological sections were
prepared for Hematoxylin & eosin (H&E), Masson’s trichrome and
Tartrate-resistant acid phosphatase (TRAP) staining. At least 8 sec-
tions per staining were selected for soft tissue histological analysis
at each time point in each group. The specimens were then exam-
ined and photographed under a high-quality microscope (Olympus
CKX41, Olympus Co. Ltd., Tokyo, Japan) and automatic digital slide
scanner (Pannoramic MIDI, 3D HISTECH, Budapest, Hungary).
2.7.5. Cross sectional analysis
The hard tissue blocks were cut to create sections of 1–1.5 mm
thickness. Cross sections were prepared by grinding the exposed
implants in a metallographic fashion with 1200, 2000 and 7000 grit
SiC paper and then polishing with a water soluble 0.5 lm diamond
slurry on microfiber. At least 8 sections were produced at each
time point in each group. The polished sections were coated with
a thin layer of gold to improve conductivity prior to imaging with
the scanning electron microscope (SEM). A SEM (Hitachi S-4800,
Japan) equipped with energy dispersive spectrometry (EDS) was
used for examining the cross sections.
2.8. Statistical analysis
Statistical analysis was performed with SPSS 18.0 software
package (SPSS Inc. Chicago. USA). Statistical significance of differ-
ence between groups was assessed by one-way analysis of variance
(ANOVA) followed by post hoc Turkey’s multiple comparison test.
P values less than 0.05 were considered statistically significant.
3. Results
3.1. Microstructures
The relative density of as-sintered pure Zn and Zn-HA compos-
ites are listed in Table 1. The densification of all materials was
higher than 90%. Addition of HA caused a decrease of the relative
density. The microstructure of pure Zn and Zn-HA composites is
shown in Fig. 1. Pure Zn consisted of particles with a large variety
of sizes and shapes while the particle size distribution was more
uniform in Zn-HA composites (Fig. 1a). HA was found homoge-
neously distributed along the Zn particle boundaries. With increas-
ing content of HA, more conglomerations were formed in particle
boundaries. The phase constituents were determined by X-ray
diffraction (Fig. 1b). Based on the diffraction pattern, Zn was iden-
tified as the major phase and the characteristic peaks of HA were
204 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214
Table 1
Compositions, SPS conditions and relative densities of pure Zn and Zn-HA composites.
Materials SPS Conditions Density (gcm
3) Relative density (%)
Pure Zn 40 MPa 7.106 (0.036) 99.524 (0.505)
Zn-1HA 380 °C 6.806 (0.019) 96.503 (0.262)
Zn-5HA 6 min 6.385 (0.022) 94.925 (0.312)
Zn-10HA 5.912 (0.039) 92.995 (0.546)
Numbers in the brackets represent the standard deviation.
Fig. 1. Microstructure of pure Zn and Zn-HA composites. (a) Optical micrographs of
pure zinc, Zn-1HA, Zn-5HA andZn-10HA composites. (b) X-ray diffraction patterns
of pure Zn and Zn-HA composites and (c) EDS analysis of area A. The inserted
diffraction pattern is the magnified XRD pattern of Zn-10HA in the 2h range of 25–
35°.
identified in the 2h range of 25–35°. Moreover, Ca and P peaks were
detected from the HA agglomerates in area A (Fig. 1c).
3.2. Mechanical properties
The mechanical properties of pure Zn and Zn-HA composites are
shown in Fig. 2. The addition of HA showed little influence on the
yield strength of pure Zn (Fig. 2a). Unfortunately, a deterioration in
tensile strength was seen with increasing HA contents. Hydroxya-
patite showed little impacts on the microhardness of Zn matrix
(Fig. 2b). The compression stress-strain curves (Fig. 2c) indicated
excellent plasticity of pure Zn with more than 90% compression
strain. However, a significant reduction in plasticity could be seen
in composites with HA contents higher than 5 wt.%.
3.3. Electrochemical measurements
The corrosion of the experimental samples in Hank’s solution
was evaluated using a short-term polarization measurement
including potentiodynamic polarization curves and Nyquist plots
(Fig. 3). The corrosion current density (Icorr) and corrosion poten-
tials (Ecorr) were determined by the Tafel extrapolation method,
and the calculated electrochemical parameters are listed in Table 2.
A significant increase of Icorr with HA contents was observed in con-
trast to pure Zn (p < 0.05). The Icorr of Zn-10HA composite was
more than 10 times as that of pure Zn (51.04 ± 1.80 lA cm
2 versus
4.90 ± 2.81 lA cm
2). The Ecorr of Zn-HA composites was decreased
as well. The polarization curves of Zn-HA composites showed a dis-
tinct features compared with that of pure Zn (Fig. 3a). Current pla-
teaus could be seen in the curves of Zn-HA composites which
indicated a passivation behavior during corrosion. The electro-
chemical impedance spectroscopy (EIS) responses of Zn-HA com-
posites characteristically appeared two semicircle-like curves
which corresponded to two time constants, mainly including two
capacitive loops (Fig. 3b). The capacitive loop at high frequency
domain was associated with the corrosion products formed on
the uniform corrosion regions on the sample surface. The capaci-
tive loop at low frequency domain was related to the interfacial
charge transfer process and electrochemical double-layer effects
at the sample/electrolyte interface. The diameters of semicircles
obtained from Zn-HA composites were smaller than that of pure
Zn, indicating a decrease in corrosion resistance.
3.4. Immersion tests
The degradation property of pure Zn and Zn-HA composites was
evaluated by immersion tests in Hank’s solution at 37 °C for 50
days (Fig. 4). The corrosion morphologies of experimental samples
after immersion are shown in Fig. S1 and Fig. 4a1. From a macro
level, pure Zn kept almost intact with a uniform corrosion mode
while corrosion pits populated with increasing HA contents. Local-
ized corrosion was dominant in Zn-HA composites and severe cor-
rosion attack was found in Zn-10HA composite sample. The SEM
images indicated a continuous and compact corrosion layer of pure
Zn. Fine precipitates distributed homogenously on the sample sur-
face. The scratches formed during polishing were still visible indi-
cating the corrosion layer was relatively thin. Similar morphology
was seen in Zn-1HA surface with some cracks showed up. An
increasing amount of corrosion products and bigger spherical pre-
cipitates were generated in Zn-5HA and Zn-10HA composites. After
removal of corrosion products, the fresh surface of pure Zn was
smooth and integral. Some corrosion pits and particle boundaries
were observed in Zn-1HA composite sample. The surface of Zn-
5HA and Zn-10HA composites were corroded severely. Lamellar
corrosion morphology was found typically in Zn matrix. Corrosion
pits distributed in Zn matrix and particle boundaries. The agglom-
eration of HA can be clearly seen in a white feature due to poor
conductivity.
The corrosion rates calculated from the weight loss was dis-
played in Fig. 4a2. Addition of HA resulted in a wide range distribu-
tion of corrosion rates compared with pure Zn. Zn-1HA composite
exhibited a slight decreased corrosion rate while addition of HA
higher than 5 wt.% accelerated the corrosion rates significantly
(P < 0.05). The corrosion rate of Zn-10HA composite was about 5
times as that of pure Zn (24.88 ± 2.33 lm/y versus 4.97 ± 1.32
lm/y). The trend of released Zn2+ concentrations in Hank’s solution
was in consistent with the results of weight loss (Fig. 4a3). The
released Ca2+ concentrations followed an opposite trend that may
due to the increasing amounts of corrosion products and precipi-
tates formed with increasing HA contents (Fig. 4a4). The evolution
of pH values during immersion is shown in Fig. 4a5. Pure Zn and
Zn-HA composites shared a similar trend of pH evolution during
immersion in Hank’s solution. A rapid increase in pH values was
seen in the first 7 days after which a relatively steady state was
achieved. The degradation of Zn-HA composites induced higher
pH values than that of pure Zn. Their pH values reached to approx-
imately 8.6 after 50 day’ immersion.
Fig. 4b shows the composition analysis of experimental speci-
mens after immersion. The cross sections revealed distinct struc-
tures of corrosion products formed on pure Zn and Zn-HA
composites (Fig. 4b1). The SEM image was composed by epoxy,
corrosion products and composite matrix. A compact, roughly 5-
lm-thick, corrosion layer was seen covering the pure Zn surface.
EDS mapping revealed Zn, O and P as the main components of cor-
rosion products. The fresh Zn surface was relatively smooth and
continuous. In contrast, irregular and non-uniform corrosion prod-
ucts were seen for Zn-10HA composite. Some rod-like products
seemed to grow from the substrate. The corrosion products mainly
consisted of Zn, O and P. Moreover, HA agglomerations rich in Ca, P
and O was visible in Zn particle boundaries. The Zn matrix beneath
 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214 205

Fig. 2. Mechanical properties of pure Zn and Zn-HA composites. (a) Compressive strength, (b) microhardness and (c) compressive stress-strain curves, *p < 0.05.

Fig. 3. Electrochemical measurements of pure Zn and Zn-HA composites in Hank’s solution. (a) Polarization curves, (b) Nyquist plots.

Table 2 extracts was 51.99 ± 8.5 at the first day and continuously
Electrochemical parameters for pure Zn and Zn-HA composites in Hank’s solution. decreased over time. According to the ISO 19003-5 [20], pure Zn
Materials Icorr (lAcm
2) Ecorr (V) Corrosion rate (mmyear
1) was cytotoxic to MC3T3-E1 cells. In contrast, significant improve-
Pure zinc 4.900 (2.810)
0.942 (0.075) 0.073 (0.042)
ments in cell viability can be observed in Zn-HA composite groups
Zn-1HA 21.076 (3.251)*
1.281 (0.037)* 0.327 (0.050)* (p < 0.05). A good biocompatibility to MC3T3-E1 cells was found
Zn-5HA 39.127 (0.661)*
1.274 (0.005)* 0.630 (0.011)* for all Zn-HA composites.
Zn-10HA 51.044 (1.803)*
1.290 (0.010)* 0.856 (0.031)*

Numbers in the brackets represent the standard deviation, *p < 0.05, compared with
pure zinc.
the corrosion products showed a jagged edge. No clear boundary
was found between HA and corrosion products.
EDS and XRD were used to further investigate the chemical
compositions of corrosion products (Fig. 4b2, b3). Zn, O and P were
major elements in corrosion products of pure Zn. With increasing
HA, the proportion of Zn decreased while O, P and Ca increased.
Moreover, the Ca/P atom ratio of corrosion products increased
from 0.54 of pure Zn to 1.41 of Zn-10HA composite. XRD spectra
of pure Zn identified ZnO and CaCO3 as main crystallized products.
As for Zn-HA composites, clear Bragg peaks of Zn3(PO4)24H2O
were identified as well. The results of EDS and XRD provided clear
evidence of a change in surface condition with different HA
contents.
3.5. In vitro tests
3.5.1. Biocompatibility
Fig. S2 illustrates the cell viability of MC3T3-E1 cells after 1, 2
and 4 days’ incubation in pure Zn and Zn-HA composite extraction
mediums. The viability of MC3T3-E1 cells cultured in pure Zn
3.5.2. Hemocompatibility
The morphologies of adhered human platelets on the experi-
mental samples are shown in Fig. 5a. Most platelets adhered on
pure Zn stayed round with no pseudopodia spreading. However,
platelets on the surface of Zn-1HA and Zn-5HA samples stretched
a small amount of pseudopodia, which may indicate a beginning
of early dendritic. As for Zn-10HA composite, the number of
adhered platelets decreased greatly and most of them showed a
round morphology. It is important to note that platelets adhered
in a homogeneous way without aggregation on all the samples.
Therefore, the thrombosis of pure Zn and Zn-HA composites was
expected to be low. The hemolysis rates of all experimental sam-
ples were far below the safe value of 5% (Fig. 5b), suggesting a
low risk of hemolysis according to ASTM F 756-08 [21].
Activated partial thromboplastin time (APTT), prothrombin
time (PT) and thrombin time (TT) are critical parameters in clinical
blood examinations and in safety evaluations of biomaterials
[22,23]. The effects of pure Zn and Zn-HA composites on APTT,
PT and TT are shown in Fig. 5c. In contrast to the control group
(normal value of human blood plasma), pure Zn and Zn-HA com-
posites induced a significant increase in PT and APTT while a
decrease in TT (p < 0.05). APTT and PT prolonged from 37.37 ±
1.76 s and 11.4 ± 0.17 s for the control to 58.15 ± 0.92 s and
13.00 ± 0.28 s for the Zn-10HA composite, respectively. TT was
206 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214

Fig. 4. In vitro immersion test. (a) Degradation behavior of pure Zn and Zn-HA composites in Hank’s solution for 50 days. (a1) Representative images of corrosion morphology
of samples before and after removal of corrosion products. (a2) Corrosion rates calculated by weight loss. (a3) Zn ion and (a4) Ca ion concentrations in Hank’s solution. (a5)
Evolution of pH values with immersion time, *p < 0.05. (b) Corrosion products analysis of experimental samples. (b1) EDS mapping of corrosion products of Zn and Zn-10HA
composite, maps of carbon, calcium, zinc, oxygen and phosphate are in green, yellow, red, blue and light blue, respectively. (b2) Elemental compositions and (b3) X-ray
diffraction patterns of corrosion products. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214 207

Fig. 5. Hemocompatibility of pure Zn and Zn-HA composites. (a) Platelets adhesion on experimental samples, (b) hemolysis percentage, (c) coagulation time including
activated partial thromboplastin time (APTT), prothrombin (PT) and thrombin time (TT) and (d) Zn ion concentrations in platelet poor plasma (PPP). Control group represents
PPP without incubation of experimental samples, *p < 0.05, compared with pure Zn, #p < 0.05, compared with control group.

shorten from 12.10 ± 0.26 for the control to 7.45 ± 0.07 for the Zn-
10HA composite. Moreover, both APTT and PT were prolonged
while TT was shortened significantly compared with pure Zn (p
< 0.05). For a better understanding of this effect, Zn2+ concentra-
tions in platelet poor plasma (PPP) are showed in Fig. 5d. The pro-
longation of APTT, PT and reduction of TT in test groups were
induced by a synchronously increase in Zn2+ concentrations in
PPP compared with control group (p < 0.05).
experimental samples. The Zn2+ concentrations and pH values in
the LB medium cultured with experimental samples for 24 h are
illustrated in Fig. 6d. The medium cultured with Ti-6Al-4V con-
tained almost no Zn2+. In contrast, the Zn2+ concentrations were
significantly increased in the medium cultured with pure Zn and
Zn-HA composites (p < 0.05). The pH values of culture medium
were ranging from 7.05 to 7.15, no statistic significant was found
between test and control groups.

3.5.3. Antibacterial property 3.6. In vivo tests

In order to examine the antibacterial effects of pure Zn and Zn-
HA composites, S. aureus were incubated for 24 h with experimen-
tal samples. After incubation, SEM was performed to investigate
the effects of materials on the bacterial morphology and mem-
brane integrity (Fig. 6a). A mass of smooth and intact S. aureus bac-
terial cells on the surface of Ti-6Al-4V sample are found which
clustered into grapelike colonies, indicating the formation of bio-
film [24]. In contrast, only a small amount of bacterial cells were
observed adhered on the surface of pure Zn and Zn-HA composites,
indicating an inhibition of bacteria adhesion and biofilm formation.
Antibacterial rates for S. aureus in the medium (Rp) and on the
specimen surface (Ra) were evaluated by the spread plate method
using Ti-6Al-4V as control (Fig. 6b, c). It can be seen that the Rp val-
ues of all the specimens reached more than 90% and the Ra values
were 80–90% as well, indicating a good antibacterial effect of
3.6.1. X-ray scanning and micro-CT analysis
Zn-5HA composite was chosen for animal tests with pure Zn as
control. Fig. 7a displays the X-ray radiographs of rat femoral con-
dyle with implants (red arrow). The images showed that pure Zn
and Zn-5HA composite implants were all located well at the sur-
gery sites without dislocation or loosening. The distinct profiles
of implants indicated good radiopacity of Zn based materials.
Moreover, no gas shadow was observed around the implants. No
significant degradation was seen for both pure Zn and Zn-5HA
composite during the entire implantation period. There was no
obvious inflammatory reactions or adverse effects around the
implants. New bone formation and osseointegration are studied
at week 4 and week 8 using micro-CT (Fig. 7b). Newly formed bone
was visualized in both groups after 4 weeks’ implantation, indicat-
208 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214

Fig. 6. Antibacterial properties of pure Zn and Zn-HA composites to S. aureus examined by the spread plate method with Ti6Al4V as control. (a) SEM morphology of S. aureus
seeded on the sample surfaces. Antibacterial rates estimated from the amount of living bacteria (b) in the medium and (c) on the sample surfaces. (d) Zn ion concentrations
and pH values in LB medium after culturing with experimental samples for 24 h, #p < 0.05, compared with control group.

Fig. 7. Micro-CT analysis of femoral condyle containing pure Zn and Zn-5HA composite implants at week 0, 4 and 8. (a) Radiographs and (b) micro-CT reconstruction images
of femoral condyle with implants. (c) 3D reconstruction models and (d) volume of metallic part of implants remained.

ing a good osteogenic property. An increase in bone volume could However, pure Zn, with tighter contact between implant and
be seen for both implants from week 4 to week 8. In contrast, the new bone tissue, exhibited better osseointegration than the com-
bone mass in Zn-5HA composite was higher than that of pure Zn. posite. The integrity was maintained for both implants during 8
 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214
weeks’ implantation. The three-dimensional analysis of implants
showed a homogeneous and slow degradation progress over the
observation period (Fig. 7c). The 3D images of Zn-5HA composite
exhibited a rougher surface morphology with more severe corro-
sion attack than pure Zn. After 8 weeks, the volume of Zn-5HA
composite dropped to 96.80 ± 0.56% compared to 98.30 ± 1.02%
on the pure Zn implant (Fig. 7d).
3.6.2. Degradation products analysis
Fig. 8 shows the morphology of the bone-implant cross sections
and the elemental mappings of bone-implant interface after 4 and
8 weeks’ implantation. Generally, pure Zn went through a macro-
scopic uniform corrosion mode without severely localized corro-
sion (Fig. 8a). No significant degradation was observed during
implantation. Only small amounts of degradation products were
visible surrounding the implant. The bone-implant interface of
pure Zn was composed by Zn matrix, degradation products, fibrous
tissue and bone. The EDS mapping illustrated that the degradation
products presented two-layer structure. The inner layer which
adjacent to the metal surface was rich in C, O and Zn, while the
outer layer consisted of more P. Bone was rich in Ca and P. The
specific atomic percentages of degradation products was shown
in Fig. S3 by select point analysis. In contrast to pure Zn, localized
corrosion attack was observed in Zn-5HA composite and its degra-
dation was more noticeable (Fig. 8b). Similar to pure Zn, the integ-
rity of the composite implant was maintained at week 8. For Zn-
5HA composite, the degradation products were accumulated
locally in a diffused way. At week 4, C, O and Zn was detected as
the predominant elements in the degradation products. In contrast,
Ca and P were detected in the outer most layer of degradation
products at week 8. Besides, intense signal of Ca and P was
detected mainly in newly formed bone and HA.
3.6.3. Histological analysis
Fig. 9 shows the tissue response to pure Zn and Zn-5HA com-
posite after 8 week’s implantation using van Gieson staining.
New bone formation could be observed surrounding pure Zn and
composite at week 4 and plenty of osteocytes were seen in the
new bone tissue. However, a thin layer of fibrous connective tissue
was seen separating the bone tissue from the implants. For Zn-5HA
composite, the localized corrosion and fast degradation rate caused
a thicker fibrous tissue layer than that of pure Zn at week 4. An
increased bone mass, extending outward from the implants, could
be observed in both implants over time, which indicated a benefi-
cial role of pure Zn and Zn-5HA composite in promoting new bone
formation. Zn-5HA composite was observed to stimulate larger
amounts of mineralized and newly formed bone than pure Zn at
week 8. In addition, the newly formed bone gradually replaced
the fibrous connective tissue and reduced the gap between com-
posite and new bone tissue. To further investigate the inflamma-
tory response, collagen formation and osteoclasts, H&E, Masson’s
trichrome and TRAP stainings are shown in Fig. 10. At week 4, both
implants were surrounded by a thin layer of connective tissue
mainly containing fibroblasts. Local infiltration of lymphocytes
and macrophages indicated a mild inflammatory response caused
by implants. Moreover, Masson’s trichrome staining illustrated
that collagen fiber build-up was evident and small islands of
osteoid were also detected surrounding the implants. After 8
weeks, inflammatory reaction with increased fibrous tissue thick-
ness was still found in pure Zn. In contrast, less inflammatory cells
and reduced fibrous tissue layer was seen surrounding the Zn-5HA
composite. Moreover, more collagen-rich bone tissue was formed
around the composite than pure Zn. Besides, no TRAP marked
osteoclasts was found in both groups at the selected time points.
209
4. Discussion
4.1. In vitro and in vivo degradation mechanism of Zn-HA composites
The basic degradation process of Zn-HA composites is similar to
that of pure Zn and the schematic graphs are shown in Fig. S5. After
immersed in Hank’s solution, Zn generally corrodes by the oxida-
tion of Zn to Zn2+ (Eq. (1)) and the electrons generated from the
anodic reaction are consumed by the corresponding cathodic reac-
tion via reduction of oxygen (Eq. (2)), which give rise to generation
of OH
. With corroding of Zn, pH value increased rapidly at early
stage as shown in Fig. 4a5. Meanwhile, the released Zn2+ reacts
with the OH
to form degradation products like oxide and hydrox-
ide (Eqs. (3)–(4)) which was found at the early stage of immersion
(Fig. S4).
Zn ! Zn2þ þ 2e
ð1Þ
O2 þ 2H2 O þ 4e
! 4OH
ð2Þ
Zn2þ þ 2OH
! ZnðOHÞ2 ð3Þ
ZnðOHÞ2 ! ZnO þ H2 O ð4Þ
Moreover, Zn(OH)2 can transform into more stable ZnO with
more exposure time [25]. However, the surface oxides thermody-
namically predicted to generate in neutral solution do not form
an effective corrosion protection barrier [26]. The Cl
in the simu-
lated body fluid readily converts ZnO and Zn(OH)2 to the soluble
ZnCl2, damaging the corrosion layer and promoting further corro-
sion. Nevertheless, a dense and compact corrosion layer mainly
consisted of Zn, O and P was observed covering the Zn matrix after
50 days’ immersion (Fig. 4b1). This can be explained by the subse-
quently formation of more thermodynamically stable phase of zinc
phosphate (Eq. (5)) [27].
3Zn2þ þ 2HPO4 2
þ 2OH
þ 2H2 O ! Zn3 ðPO4 Þ2  4H2 O ð5Þ
The absence of zinc phosphate in the XRD pattern (Fig. 4b3) of pure
Zn may cause by its amorphous structure [28]. Zinc phosphate was
reported as major degradation products both in vitro and in vivo
[27,29–31]. Therefore, ZnO and Zn(OH)2 are able to be converted
to zinc phosphate depending on the shift in the precipitation disso-
lution equilibrium. The zinc phosphate contained layer is protec-
tive and remains intact with immersion time, leading to a
relative stable pH in Hank’s solution at late stage. As a result, pure
Zn showed little degradation throughout the immersion time. The
incorporation of HA into Zn matrix results in a great influence on
the microstructure of pure Zn. Consequently, a difference in degra-
dation behavior between pure Zn and Zn-HA composites can be
expected. HA in the composites plays biphasic roles during degra-
dation: On the one hand, HA is characterized by a poor conductiv-
ity and low solubility under physiological condition. Thus, the
distribution of HA along the particle boundary creates a network
structure that impedes the charge transfer among Zn particles. As
a results, addition of HA improves the corrosion resistance of Zn
matrix. On the other hand, the relatively low sintering temperature
and pressure and lack of interface reaction between Zn and HA
during SPS process contribute to the increased porosity with HA
contents [32]. Therefore, pores and voids which form in the inter-
face of Zn particles and HA agglomerates promote the penetration
of corrosive fluid in the composite matrix. This can be proved by
the decreased relative density of Zn-HA composites (Table 1) and
cracks in the particle boundary of samples after removal of corro-
sion products (Fig. 4a1). Moreover, more sites that are vulnerable
to corrosion attack are generated due to the physical presence of
HA in the particle boundary [33]. These factors lead to the acceler-
210 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214

Fig. 8. SEM images of the cross sections. (a) Pure Zn and (b) Zn-5HA composite at week 4 and week 8 with magnified images of yellow rectangles and corresponding
elemental distribution of view in images. (Carbon (green), oxygen (purple), zinc (red), phosphate (light blue), calcium (yellow)). (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)

ated corrosion of Zn-HA composites. In this way, the degradation
rates of Zn-HA composites are adjustable by tuning the volume
fraction of HA. In contrast to pure Zn, Zn-1HA composite showed
a slight decreased degradation rate while addition of 5 wt.% and
10 wt.% HA accelerated the degradation significantly. As the degra-
dation proceeds, HA agglomerates play a positive role in sponta-
neously absorbing and inducing the accelerated deposition of
Ca2+ and PO3

4 in the solution, thereby the compact Ca, P contained
layer which can slow down the corrosion effectively forms on the
sample surface [34–37]. A significant reduction in Ca2+ concentra-
tion with increased HA contents was seen in the Hank’s solution
after immersion (Fig. 4a4). Meanwhile, Zn-HA composites showed
a corresponding increase in corrosion layer thickness (Fig. 4). The
formation of this protective layer can retard the corrosion of com-
posites. As a result, a relatively stable stage can be expected in the
evolution of pH at late period (Fig. 4a5). In contrast to pure Zn, the
apparent compositional nature of degradation products began to
change after adding HA. Elements including O, P and Ca accounted
for more proportions of degradation products while percentage of
Zn decreased with more HA contents. Moreover, the Ca/P atom
ratio of degradation products increased from 0.54 of pure Zn to
1.41 of Zn-10HA composite, which is near the Ca/P ratio of HA
(HA, Ca/P = 1.6). Previous studies have reported that HA in the
composites could act as the hydroxyapatite nuclei and stimulated
 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214 211

Fig. 9. Histological characterization of hard tissue sections at implant sites. Van Gieson staining of (a) pure Zn and (b) Zn-5HA composite at week 4 and 8. The red rectangles
correspond to the magnified bone-implant interface. The red triangle indicates newly formed bone. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

the growth of hydroxyapatite, which is beneficial to new bone
growth [34,36,38]. However, XRD is hard to confirm the newly
formed HA after immersion due to the interference of HA phase.
Diffraction pattern of Zn-HA composites identified a mixture of
zinc oxide, zinc phosphate and calcium carbonate as degradation
products (Fig. 4b3).
To further investigate the degradation behavior of Zn-HA com-
posites in vivo, pure Zn and Zn-5HA composite were implanted in
the femoral condyle of rats. Micro-CT indicated that little degrada-
tion of pure Zn took place throughout the implantation time
(Fig. 7). Moreover, the degradation proceeded in a relatively uni-
form mode. However, the compact and protective corrosion layer
failed to form under in vivo conditions (Fig. 8). In this respect,
the slow degradation rate seems to be contradicted with the
incomplete corrosion layer. This can be explained by the availabil-
ity of oxygen in the surrounding tissue, which influences the
degradation rates of Zn-based implants in vivo. The underlying cor-
rosion reaction proceeds as fast as dissolved oxygen reaches the
metal surface. Nevertheless, the availability of oxygen is limited
in the surrounding tissue of implants investigated here (34
mmHg) [39]. As a result, the cathodic oxygen reduction reaction
is inhibited which leads to the slow degradation rates of samples.
Similar results have been reported in a 52-week in vivo study of
Fe-based alloys implanted in femoral mid-diaphyseal region of rats
[40]. The limited availability of oxygen is a critical factor causing
the low degradation rates of Fe-based implants. In our previous
study, we found a discrepancy in degradation rates of Zn stents
in blood fluid and neointima, which partly due to the difference
of oxygen partial pressure in different degradation environments
[40]. In contrast to pure Zn, addition of HA induced accelerated
degradation and a localized corrosion mode (Figs. 7 and 8). Firstly,
corrosion attack preferentially happens in the interface of Zn par-
ticles and HA agglomerates. Then, the metallic parts are gradually
corroded and replaced by degradation products. The voluminous
212 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214

Fig. 10. Histological characterization of soft tissue sections at implant sites. H&E, Masson’s trichrome and TRAP staining of (a) pure Zn and (b) Zn-5HA composite after 4 and 8
weeks’ implantation. The red, light blue and black rectangles correspond to the inserted magnified areas in H&E, Masson’s trichrome and TRAP staining, respectively.

corrosion products act as wedges along the interface allowing the
corrosive fluid to penetrate inside and keep reacting with the fresh
matrix [41]. Similar to that of pure Zn, the dense and integral cor-
rosion layer formed in vitro is absent under in vivo conditions,
which facilitates the continuous degradation of Zn-HA composites.
It is worth to mention that both pure Zn and Zn-HA composites are
able to maintain the mechanical integrity for more than 8 weeks
which is critical important for bone healing process [7]. Analysis
of the degradation products can help us understand the in vivo cor-
rosion process of implants. The EDS investigation revealed that the
degradation products of both pure Zn and Zn-5HA composite were
characterized by layered structure (Figs. 8, S3). The inner layer
adjacent to the matrix mainly consisted of oxidized constituents
of the material, i.e. C, O and Zn. On top, there is an outer layer that,
besides C, O and Zn, also contained elements that originate from
the surrounding tissue, i.e. P, Ca. Several works have reported the
formation of zinc oxide, zinc carbonate and Ca/P phase as major
degradation products of pure Zn wire in abdominal aorta of rats
[2,42], but we fail to find any references related to the in vivo
degradation of Zn based materials in bone environments. In this
study, zinc oxide is believed to be one of the main components
of degradation products. Besides, Ca/P phase could generate in
the outer region induced by the release of Ca and P ions from HA
or local tissues and a local rise in the pH value. However, the for-
mation of zinc carbonate is hard to confirm because the contami-
nation of carbon during sample fabrication. The discrepancy in
morphology and composition of degradation products found
in vitro and in vivo suggests that the degradation of the Zn-based
composites is greatly influenced by the surrounding environments.
The in vivo degradation process is more complex due to the expo-
sure of materials to an environment rich in a diversity of ions and
organic substances such as proteins and cells.
4.2. Biocompatibility
Formation of new bone was visualized by micro-CT, SEM and
histological analysis in both implants at week 4 (Figs. 7–10). How-
ever, both implants showed a fibrotic and collagenous encapsula-
tion response separating the new bone tissue and materials. In
contrast, Zn-5HA composite was surrounded by a thicker fibrous
connective tissue than pure Zn. An increased bone mass could be
seen with prolonged implantation time in both implants. In addi-
tion, Zn-5HA composite showed a more pronounced effect in stim-
ulating new bone formation at week 8. And fibrous layer was
 H. Yang et al. / Acta Biomaterialia 71 (2018) 200–214
gradually replaced by new bone tissue over time. Nevertheless, the
lack of direct bone bonding to the implants at week 8 indicates a
delayed osseointegration in both pure Zn and composite. Gener-
ally, for biodegradable materials, the tissue response is closely
related to the degradation process of materials. The in vivo degra-
dation of pure Zn creates solid products like oxide, hydroxide and
Ca/P compound and releases chemical species like Zn ions and
hydroxide ions. Apart from this, the disintegration and dissolution
of hydroxyapatite generates Ca and P ions from Zn-HA composites
[43–45]. Zn, as an essential element for basic biological function,
shows biphasic effects on new bone formation. Previous studies
have reported enhanced mineralization of extracellular matrix
and human bone marrow mesenchymal cells (hMSC) osteogenic
differentiation and increased expression of bone-related genes
including alkaline phosphatase, collagen I and osteopontin when
hMSC were cultured with Zn or Zn ions [46–48]. Zn also exhibited
an inhibitory effect on osteoclast differentiation [49,50]. In vivo
animal studies showed efficient promotion on new bone formation
and bone contact induced by Zn incorporated materials [48,51,52].
However, excessive release of Zn ions can induce a cytotoxicity and
DNA damage on human osteoblast cells and may even cause bone
resorption in vivo [10,51,53]. Hydroxyapatite is one of the fre-
quently used bioceramics for bone tissue reconstitution. It pos-
sesses excellent biocompatibility with hard tissue, high
osteoconductivity and bioactivity [54]. Hydroxyapatite scaffold
has shown an enhanced effect on the proliferation rate and osteo-
genic differentiation of hMSC [55]. Moreover, promotion of new
bone formation by hydroxyapatite was confirmed in animal and
clinical tests [43,56]. As a result, HA is a favorable choice to incor-
porate into matrix like magnesium or polymer in order to increase
their biocompatibility and bioactivity [18,57,58]. In the present
study, the in vitro cell test showed a great toxicity of pure Zn on
MC3T3-E1 cells while addition of HA improved the cytocompatibil-
ity significantly (Fig. S2). In comparison, the effects of HA are more
complicated under in vivo conditions. In the early stage of implan-
tation, an increased amount of fluid accumulates in the cancellous
tissue as a result of tissue trauma. Meanwhile, a haematoma is
generated and consisted of cells from blood and bone marrow, fol-
lowed by an inflammatory response [59]. Therefore, a relatively
rapid degradation with large amounts of Zn ions released from
implants is expected at this time. As a result, the inhibitory effect
of Zn ions is predominant and a decreased Zn ion gradient can form
with increasing distance from the implant. In the vicinity of
implant, Zn ion concentration is too high to allow new bone forma-
tion. Consequently, a fibrous connective tissue instead of new bone
forms, which causes delayed osseointegration. The addition of HA
induced accelerated degradation and localized corrosion which
led to a thicker fibrous tissue layer than pure Zn (Figs. 8 and 9).
As bone healing progresses, the haematoma is replaced by a
fibrin-rich granulation tissue, followed by formation of cartilagi-
nous tissue and mineralization. Less fluid and reduced inflamma-
tion accompanied this process. As a result, the degradation of
implants slows down and less Zn ions are released over time. Then,
the beneficial role of Zn ions in stimulating new bone formation
becomes more prevailed. In contrast to pure Zn, Zn-5HA composite
exhibited a better performance in osteogenesis at week 8, which
induced by the combined promotion effects of Zn and HA. Despite
the larger release of Zn ions at early stage, Zn-HA composites pro-
duced superior biological responses in bone growth than pure Zn
with prolonged implantation time. According to the degradation
rates, the released Zn for pure Zn and Zn-5HA composite was about
0.035 and 0.059 mg/day, both of which are far below the daily
allowance of zinc (15 mg/day) [2]. Therefore, the biosafety of Zn-
HA composites will be guaranteed.
213
5. Conclusions
Incorporation of HA into pure Zn is an effective approach to
adjust its degradation rate and improve its biocompatibility both
in vitro and in vivo. A wide range of degradation rates can be
achieved by changing the concentrations of HA. Cell test indicated
a significant better cytocompatibility of Zn-HA composites. In vivo
tests showed that addition of HA resulted in a better performance
in osteogenesis with prolonged implantation time. Further studies
will investigate the combinations of Zn alloys with better mechan-
ical performance as matrix and more bioactive reinforcements.
Moreover, a better interface bonding between matrix and addi-
tions is necessary to improve the corrosion uniformity. This sys-
tematic study suggests that Zn based MMC can be a promising
strategy to improve the performance of pure Zn for applications
in orthopedic implants.
Acknowledgements
This work was supported by the National Key Research and
Development Program of China (Grant No. 2016YFC1102402),
National Natural Science Foundation of China (Grant No.
51431002), NSFC/RGC Joint Research Scheme (Grant No.
51361165101 and 5161101031) and NSFC-RFBR Cooperation Pro-
ject (Grant No. 51611130054). Hongtao Yang and Xinhua Qu con-
tributed equally to this study.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.actbio.2018.03.007.
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