Accepted Manuscript

Title: In vivo study of microarc oxidation coated

biodegradable magnesium plate to heal bone fracture defect of
3 mm width

Authors: Y.F. Wu, Y.M. Wang, Y.B. Jing, J.P. Zhuang, J.L.
Yan, Z.K. Shao, M.S. Jin, C.J. Wu, Y. Zhou

PII: S0927-7765(17)30391-0
DOI: http://dx.doi.org/doi:10.1016/j.colsurfb.2017.06.031
Reference: COLSUB 8641

To appear in: Colloids and Surfaces B: Biointerfaces

Received date: 15-3-2017

Revised date: 12-6-2017
Accepted date: 21-6-2017

Please cite this article as: Y.F.Wu, Y.M.Wang, Y.B.Jing, J.P.Zhuang, J.L.Yan, Z.K.Shao,
M.S.Jin, C.J.Wu, Y.Zhou, In vivo study of microarc oxidation coated biodegradable
magnesium plate to heal bone fracture defect of 3mm width, Colloids and Surfaces B:
Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.06.031

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 In vivo study of microarc oxidation coated biodegradable

magnesium plate to heal bone fracture defect of 3mm width

Y.F. Wua, Y.M.Wanga, *,Y.B. Jingb, J.P. Zhuangb, J.L. Yanb, Z.K. Shaoc, M.S. Jinc, C.J. Wua, Y. Zhoua

aInstitute for Advanced Ceramics, Harbin Institute of Technology, Harbin 150001, China
bThe Second Affiliated Hospital, Harbin Medical University, Harbin 150001, China
The First Affiliated Hospital, Harbin Medical University, Harbin 150001, China
c

 Corresponding author. Tel.: +86-451-86402040-8403; fax: +86-451-86414291.

E-mail addresses: wangyaming@hit.edu.cn (Y.M. Wang)

Graphical abstract

Highlights
 10 and 20 thick coatings containing Ca, P, Si were obtained on Mg plates by MAO.

 Fracture model of 3mm width defect was adopted to evaluate degradation and healing.

 The releasing Mg2+ by the degradation of implants promoted bone fracture healing.

 Mg degradation and bone fracture healing can be tailored by MAO coating.

Abstract
Microarc oxidation (MAO) coated magnesium (Mg) with improved corrosion resistance appeal increasing
interests as a revolutionary biodegradable metal for fractured bone fixing implants application. However, the in
vivo corrosion degradation of the implants and bone healing response are not well understood, which is highly

11
required in clinic. In the present work, 10 μm and 20 μm thick biocompatible MAO coatings mainly composed of
MgO, Mg2SiO4, CaSiO3 and Mg3(PO4)2 phases were fabricated on AZ31 magnesium alloy. The electrochemical tests
indicated an improved corrosion resistance of magnesium by the MAO coatings. The 10 μm and 20 μm coated
and uncoated magnesium plates were separately implanted into the radius bone fracture site of adult New
Zealand white rabbits using a 3 mm width bone fracture detect model to investigate the magnesium implants
degradation and uninhibited bone healing. Taking advantage of the good biocompatibility of the MAO coatings,
no adverse effects were detected through the blood test and histological examination. The implantation groups of
coated and uncoated magnesium plates were both observed the promoting effect of bone fracture healing
compared with the simple fracture group without implant. The releasing Mg2+ by the degradation of implants into
the fracture site improved the bone fracture healing, which is attributed to the magnesium promoting
CGRP-mediated osteogenic differentiation. Mg degradation and bone fracture healing promoting must be tailored
by microarc oxidation coating with different thickness for potential clinic application.

Keywords: Magnesium; Biodegradation; In vivo study; Fracture healing

1. Introduction
Fracture is a common trauma with a long healing period. Among the fractures, approximately one third of
which require internal fixation devices to assist and promote healing [1]. Traditional clinical applications are
mainly used of stainless steel, titanium alloys or cobalt-based alloys [2]. However, massive difference of
mechanical properties such as the elastic modulus between the traditional implants and the natural bone will
induce the stress shielding effect [3] which caused interference with skeletal growth and may induce secondary
bone fracture [4] . Therefore, these materials may necessitate invasive removal surgeries, causing an economic
and physical burden to the patients, and prolonging the cure period [5, 6]. To mitigate these concerns,
biodegradable materials have been developed, which are mainly involved with restorable polymers and ceramics.
Nevertheless, poor mechanical strength of the polymers and brittleness of the ceramics often limit them as viable
options for load-bearing devices [2]. For these reasons, there remains a need to develop biodegradable materials
with proper mechanical properties, good biodegradability or low biotoxicity, and suitable degradation properties
matching the tissue healing [7], which encourages scientists to explore the possibility of using various materials,
such as magnesium [8-10].
Magnesium and its alloys, as a kind of biodegradable metal, can be degraded and then discharged through
metabolism when complete its service time, which avoided the second surgery to take out the implants [11, 12].
Similar elastic modulus between magnesium (40-45 GPa) and natural bone (10-40 GPa) minimizes the stress
shielding [13, 14]. Magnesium is a macro element of human body that plays an important role in metabolism [15],
so that a moderate release of Mg2+ ion will not cause any toxic or side effects [16]. Moreover, some studies have
investigated the biocompatibility and osteoconductivity of magnesium alloys, in which high mineral apposition
rates and increased bone mass and mineral density surrounding magnesium implants in bone were detected
[17-20]. The results imply that Mg2+ may facilitate new bone formation.
The biodegradability and the similar mechanical properties with human bone suggest that magnesium and
its alloys have the potential to be good mechanical load-bearing devices [7, 21]. However, high corrosion
susceptibility [22, 23] of magnesium in physiological environment limits its development. With the rapid
corrosion degradation of magnesium, the device may lose efficacy as the decrease of mechanical properties
before complete healing of tissue [24]. Meanwhile, the release of Mg2+ ion and the excessive hydrogen formation
may be over load for human body metabolism [25]. In order to resolve the key issues on earlier mechanical failure
due to the rapid degradation rate, alloying [21] and surface modification [7, 26, 27] are achieved to control the
degradation rate. For surface modification, some incorporated elements in the coatings reveal the positive

12
impacts. Bala SP et al. [28] reported a Ca-P contained surface coating showed excellent corrosion resistance.
Valerio P et al. [29] showed that Ca, Si elements promote the nucleation and growth of CHAp which promotes the
proliferation and differentiation of osteoblast. Hala Zreiqat et al. [30] demonstrated that the releasing of Ca, Si,
and Mg from a CaMgSi2O6 coated stent showed excellent bioactivities.
As load-bearing implants, the magnesium devices work in vivo, reacting with the surrounding tissues and
thus suffering from continuously friction and extrude stress, which is of great challenges to the stability of
protecting coatings. Microarc oxidation (MAO) is feasible to fabricate ceramic coatings on magnesium and its
alloys with high adhesion strength to the substrate and excellent wear resistance [31], thus providing a long-term
stable protecting ability avoiding peeling and abrasion by the mechanical movement. Meanwhile, the corrosion
resistance of the magnesium based implants can be also improved by the protection of MAO coatings [32]. More
importantly, the composition and structure of the MAO coatings can be tailored by adjusting the electrolyte and
electrical parameters so that proper temporary coatings could be conducted [33, 34]. Xiao et. al [35] investigated
the in vitro degradation behavior of a Sr-containing phosphate-based MAO coating on ZK60 magnesium alloy and
they found that the MAO coatings exhibited good corrosion resistance and excellent long-term protective ability.
Furthermore, the blood compatibility of the magnesium implants was improved prominently by MAO coatings
[36]. Herein, we fabricated a Ca, P, Si contained MAO coatings on magnesium substrate for bone fracture fixation
application, utilizing the advantages of the good corrosion resistance property, good mechanical properties of
MAO coatings and the biocompatibility of Ca, Si, P combined compounds.
The in vivo degradation behaviors of magnesium based implants [37-39] are rather critical for clinical
application. Different from the permanent implants such as stainless steel, the interface between the Mg-based
implants and biological environment is dynamic [40]. Importantly, although a large number of animal attempts
have been conducted to investigate the biosafety and bio-efficacy of magnesium based implants, most of these
animal models were oversimplified by just inserting Mg based devices in the femoral cavity or shaft without
establishing clinical indication-oriented surgeries [41]. However, the in vivo surroundings are varied in different
implantation sites, owing to the behavior of surrounding host cell and the ability of blood supply, which caused
different corrosion rate and tissue response in vivo. As a result, these may cause discrepancies in healing
outcomes between in vivo experimental studies and clinical trials [41]. Hence, for clinical requirements, various
implantation models should be conducted to investigate the material degradation and tissue response particularly.
Thus, for fracture fixation devices development, implantation using a proper animal fracture model is significantly
important. In this study, we employed a radius fracture model of 3 mm width bone defects using New Zealand
white rabbits. The MAO coated/uncoated magnesium implants were inserted adjoin the fracture site to imitate
fracture trauma. 3 mm width bone defects are employed to delay the naturally bone fracture healing period.
Herein, the 3 mm width bone fracture implantation model was employed, aiming at answering the following
questions: (i) How fast do the naked and MAO coated magnesium implants degrade in vivo? (ii) What is the effect
of magnesium alloy degradation on bone healing response and do the MAO coated magnetism adjust the
degradation rate successfully in this model? (iii) To what extent does the hydrogen gas gather in this model and
does the gas influence the surrounding tissues?
To summarize the resolving strategy, we fabricated a microarc oxidation coatings contained Ca, P, Si on AZ31
magnesium plate to investigate the in vivo corrosion degradation of the implants and bone fracture healing
response using a 3 mm width bone fracture defect model. Different thickness of the MAO coatings was fabricated
to control the corrosion degradation rate. The constituent and structure of the coatings, the degradation of the
implants, the biosecurity of the implants and the bone response to different degradation rates of the implants
were focused.

2. Materials and methods

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2.1 Samples preparation
Commercial AZ31 magnesium alloy (Al 3.0–3.2%, Zn 0.8–1.2%, Mn 0.2%, balance Mg) was used as the
substrate for MAO treatment. The samples were machined into plates with the size of 3 mm × 30 mm × 2 mm.
The samples were polished using 400#, 800# and 1200# abrasive paper, and cleaned in distilled water followed by
ultrasonic treatment in acetone.
The Mg plates were used as anodes and a stainless steel plate was used as a cathode in the electrolytic bath.
The electrolyte was prepared by the dissolution of reagent-grade chemicals of Ca(H2PO4)2 (10 g L-1), Na2SiO3 (15 g
L-1) and NaOH (10 g L-1) into deionized water. A 65 kW MAO power device was adopted to provide the oxidizing
energy and the electrical parameters were fixed as voltage 400 V, frequency 600 Hz and duty cycle 8%. The
temperature of electrolyte was kept lower than 40 oC by a cooling system. 10 μm and 20 μm thick MAO coatings
were fabricated on magnesium samples by oxidation duration periods for 5 min and 10 min. For convenience, the
uncoated magnesium, 10 μm coated and 20 μm coated magnesium samples were named as U-Mg plate, CPS10
plate and CPS20 plate respectively.
2.2 Coating characterization
Scanning electron microscopy (SEM, Helios Nanolab 600i, FEI Co., America) was used to observe the surface
morphologies of the samples and determine the thickness of the coatings before implantation. The phase
composition of the MAO coatings was analyzed by X-ray (XRD: Philips X’Pert) using a Cu Kα (radiation at a grazing
angle of 3°).
The potentiodynamic measurements were used to evaluate the corrosion resistance of MAO coatings. A
CHI604C electrochemistry workstation was used. All electrochemical measurements were conducted using a
conventional three-electrode electrochemical cell with the samples as the working electrodes, a platinum plate as
the auxiliary electrode and a saturated calomel electrode (SCE) as the reference. The samples were allowed to
stabilize at their open circuit potential for 30 min before the experiment started. The potentiodynamic
measurements were conducted at a sweep rate of 0.01 mV s-1 from -2.0 V to -1.0 V at 25 oC in simulated body
fluid (SBF, 8.035 g L-1 NaCl, 0.355 g L-1 NaHCO3, 0.225 g L-1 KCl, 0.231 g L-1 K2HPO4·3H2O, 0.311 g L-1 MgCl·6H2O, 3.9
ml L-1 10 mol L-1 HCl, 0.292 g L-1 CaCl2, 0.072 g L-1 Na2SO4, 6.118 g L-1 Tris).
2.3 Implantation
21 healthy, mature, male/female New Zealand white rabbits (weight: 2.5-3.0 Kg) were chose to investigate
the effects of MAO coated magnesium plates on fracture healing behaviors. All aspects of the animal care
complied with the Animal Welfare Act and the recommendations by the NIHPHS Guide for the Care and Use of
Laboratory animals. The animals were randomly divided into 4 groups for the implant of U-Mg plate, CPS10 plate,
CPS20 plate, and the last group was the control group with simple fracture treatment but no samples implanted.
All of the U-Mg group, CPS10 group and CPS20 group were divided into 2 subgroups with 3 rabbits each as we
retrieved the plates at 4 and 8 weeks after implantation. The fracture surgery was conducted on the radius of
both sides of the animals. Similar to the other groups, the control group is named S-F group (simple fracture
group).
The implanted model for fracture healing is showed in Fig. 1. A defect with 3 mm width was made in the
radius of both sides of the rabbits. 3 mm wide defect is set in a safe range in which the defect can be healing by
the rabbit itself. This model is different from the model of plate and screw [42, 43]. Because the radius and ulna
of the rabbit forelimb can support each other, it’s no necessary to fix the fracture bone and the plate.
2 mg kg-1 10% chloral hydrate solution was injected into the ear vein of the rabbits. After the eye
conjunctiva reflection disappeared, the rabbits were fixed on the operation table protractedly, followed by
preoperative hair removal, regular disinfected and draped to reduce surgical site infection. The fracture model
was shown as Fig. 1. The plate was implanted symmetrically on the surface of the fracture site and then fixed by

14
surgical suture and the surgery legs were fixed by the medical bandages. After implantation, each rabbit was
injected with antibiotic of gentamicin by a dose of 1 mL a day for 3 days. The normal dietary intake by rabbit was
performed and the suture was removed after implantation for 10 days.
After implantation for 4 and 8 weeks, the rabbits were sacrificed by injection of atmosphere into the ear
vein. Then the radii were detached from the rabbits immediately for the further treatment and the remained
organs were treated safely.
The heart blood from the animals was collected to examine the Mg2+ for the evaluation of the biosecurity
status after implantation for 4 and 8 weeks.
2.4 Radiographic evaluation
After implantation for 4 and 8 weeks, the X-ray images of U-Mg plates, CPS10 plates, CPS20 plates and
simple fracture of both sides of the legs were taken by an X-ray digital photographic technique (XPLORER1600,
IDC Co., Canada) with the parameter of 55 KV, 0.12 s, 50 mA to examine the implantation status.
2.5 Histological analysis
(1) Decalcified bone biopsy
The taken out radii were immersed into 10% formaldehyde solution for 24 h, rinsed by running water. The
samples were dehydrated by 80% alcohol for 24 h and 5% nitric acid aqueous solution for 5 D. The 5% nitric acid
aqueous solution was refreshed every 24 h.
(2) Conventional biopsy
The taken out kidney tissue and heart tissue of the animals were immersed into 10% formaldehyde solution
for 24 h, rinsed by running water for 30 min, by ethanol-formaldehyde solution for 120 min. Then the samples
were dehydrated by alcohol with different concentrations of 80% (80 min), 95% (80 min), 95% (80 min), 100% (50
min), 100% (50 min) and 100% (50 min).
(3) Tissue sectioning and HE staining
The samples were taken the transparent treatment by xylene for 60 min while the xylene was refreshed
every 20 min, and then embedded by 50° paraffin (60 min), 58° paraffin (60 min) and 58° paraffin (60 min). The
embedded samples were sliced to flakes with thickness of 100 μm by a sawing slicing machine (Leica 1600), then
fixed on the slide and baked 20 min.
The sections were dewaxed by xylene, then immersed into the alcohol with different concentrations of 100%,
100%, 95%, 90%, 85%, rinsed by distilled water, immersed into Hematoxylin dyeing (10 min), rinsed, immersed
into the mixed solution of 0.5% HCl and 70% alcohol till the cell nucleus turned amaranth and the cytoplasm
colorless, rinsed till the cell nucleus turned blue, immersed into 0.5% Eosin solution (3 min), rinsed, dehydrated
by alcohol with different concentrations of 85%, 90%, 95%, 95%, 100%, 100%, immersed into xylene again for 60
min while the xylene was refreshed every 20 min. The samples were observed by microscope.

3. Results
3.1 Coating characterization
Fig. 2(a1), Fig. 2(a2), Fig. 2(b1) and Fig. 2(b2) present the surface and cross-sectional morphologies of CPS10
and CPS20 coated magnesium plates with different oxidation time. In the process of microarc oxidation, when the
applied voltage exceeds a certain threshold, dielectric breakdown occurs in some scattered weak regions across
the insulating layers, accompanied by the phenomenon of spark discharge [44]. The spark discharge, gathering on
the surface of the metal, induce the growth of MAO coatings and results in the numerous micropores on the
surface as shown in Fig. 2(a1) and Fig. 2(b1). As the oxidation time extending from 5 min to 10 min, the size of
micropores changed apparently, on which a uniform pore diameter size of about 2-3 μm for 5 min turned to
toughness surface with the pore diameter size ranging from 2-10 μm for 10 min. In addition, with the increasing

15
of oxidation time, the number of micropores decreased obviously. Actually, to the essence of spark discharge,
each sparking event is corresponding to a discharge channel penetrating the coatings from the substrate to the
electrolyte. Thus, the increased size and decreased number of micropores in the discharge process are mainly
owing to that the constant energy is concentrated to breakdown the formative increasing MAO coatings under a
fixed voltage of 400 V with the oxidation time prolonging, which result in less discharge channels and larger pores.
Meanwhile, it can be seen in Fig. 2(a2) and Fig.2(b2), the thickness of MAO coatings increased approximately
from 10 μm to 20 μm accompanied by the increased oxidation time. From the cross-sectional morphologies of
the coatings, the interior pores exist both in the 10 μm and 20 μm coatings, which is mainly attributed to rapid
cooling down of the reaction products and blocking the discharge channels.
In the process of microarc oxidation, the formative coatings undergo a severe cyclic dynamic transforming,
involved with the coating dissolution, melting or sputtering, and then some complex chemical reactions occur to
produce various oxidation products mainly in the position of discharge channels. Under the effect of electrical
field, Mg from the substrate loses electron and turn to Mg2+, while OH-, SiO32-, PO43- from the electrolyte are
drown into the discharging area through discharging channels. Meanwhile, Ca 2+, as well as the OH-, SiO32-, PO43-
are drown into the discharging area by the effect of solution diffusion. Therefore, MgO, CaSiO 3 and Mg2SiO4
phases etc. deposit as the microarc oxidation products by the following possible reactions:
Mg2+ + 2OH- → Mg(OH)2 (1)
Mg(OH)2 → MgO + H2O (2)
2Mg2+ + SiO32- + 2OH- → Mg2SiO4 + H2O (3)
Ca2+ + SiO32- → CaSiO3 (4)
H2PO4- + OH-→ HPO42- + H2O (5)
HPO42- + OH- → PO43- + H2O (6)
3Mg2+ + 2PO43- → Mg3(PO4)2 (7)
As expected, the coating contains MgO (JCPDS #89-7746), Mg2SiO4 (JCPDS #75-1448), CaSiO3 (JCPDS
#73-1110) and Mg3(PO4)2 (JCPDS #75-1491) phases which is shown in Fig. 2(c). In addition, the Mg2SiO4, CaSiO3
and Mg3(PO4)2 phases will be helpful to enhance the biocompatibility of the coatings [45].

In a typical polarization curve, lower corrosion current density, positive corrosion potential and higher
polarization resistance correspond to lower corrosion rate and better corrosion resistance of the coatings [46].
The obtained potentiodynamic polarization curves and related parameters including the corrosion potentials
(Ucorr), corrosion current densities (icorr) and polarization resistance (Rccor) are shown in Fig. 2(d) and Table 1,
respectively.
From Fig. 2(d) and Table 1, the corrosion potential of the U-Mg plate is as low as -1.778 V, while the 10 μm
and 20 μm thick MAO coatings enable the corrosion potential increase up to -1.532 V and -1.391 V. Meanwhile,
the corrosion current decreases by two orders of magnitude, from 3.708×10-5 A cm-2 of the U-Mg plate to
2.133×10-7 A cm-2 of the CPS10 plate and 2.482×10-7 A cm-2 of the CPS20 plate. The results illustrate that the MAO
coatings can enhance the corrosion resistance of the Mg alloy effectively, which implying that CPS10 and CPS20
coated plates can maintain the mechanical integrity for a longer time than the U-Mg plate.
3.2 Bone response
Fig. 3 presents the bone tissue detections of the implanted animals with U-Mg, CPS10 and CPS20 plates
after 4 and 8 weeks postoperatively. 4 weeks postoperatively, the continuous trabecular bone appeared but was
in a disordered arrangement state. The connectivity of the bone trabecular of U-Mg group was better than the
other groups. 8 weeks postoperatively, the trabecular bone was more mature and the maturity of U-Mg group
was higher than CPS10 and CPS20 group.

16
 Fig. 4 shows the bone fracture healing process of different period. At 4 weeks after surgery, bone callus was
detected at fracture site. There was more bone callus grown in the three implanted groups than the control group
with no implantation. Among the three implanted groups, U-Mg group showed the best bone fracture healing
rate as the bone callus grew more, followed by CPS10 group. At 8 weeks after surgery, the bone fracture healing
of three implanted groups proved better than the control group. Among them, the fracture healing of U-Mg
group exhibited best, and the medullary cavity of it had been partially reopened. The taken out implants were
also observed to evaluate the in vivo degradation and corrosion resistance. The substrate magnesium plates
showed the fastest corrosion rate followed by CPS10 plate and CPS20 plate. In the first 4 weeks of implantation,
some corrosion pits were observed on the U-Mg plates and corrosion products deposited on the surface of the
plates. During 8 weeks after surgery, the corrosion rate accelerated and defects on the surface of the plates
increased. However, 8 weeks after implantation, the plates still kept complete shape.

.
3.3 Biosecurity of the implants
Fig. 5 shows Mg2+ hematology test results in different implantation time of 4 groups of rabbits after surgery.
The serum Mg2+ concentration of the S-F group maintained the similar level after 4 and 8 weeks’ surgery.
Although the serum Mg2+ concentration of the implanted groups were all higher than S-F group, the detected
serum Mg2+ concentration was below 2.0 mmol L-1, which is within the normal range of physical magnesium level
[47]. Comparing all groups, the MAO coated magnesium implants maintained a lower level than the bare
magnesium, indicating that in vivo degradation of the MAO coated magnesium implant did not induce a great
increase of magnesium ions.
To determine the health status of the implanted animals, the hematology test and pathological examination
were carried out during the implantation. The renal histological detections of the implanted rabbits with U-Mg,
CPS10 and CPS20 plates for 4 and 8 weeks are shown in Fig. 6(a). It can be seen from the images that the volume
and shape of glomerular as well as the shape of renal tubular are in a normal condition while no obvious edema
and hyperplasia of endothelial cells can be seen. The cardiac histological detections of the implanted animals with
the same condition can be observed in Fig. 6(b) . The images indicate that the neat myocardial fibers are all neatly
arranged and the volume and shape of the myocardial cell are in a normal condition. No cell proliferation or cell
necrosis can be found. Different conditions (the different implants and the implant time) show no differences in
the results described above.

4. Discussion
4.1 Biosecurity of the implants
In the present study, magnesium implants with and without MAO coatings were implanted into a fracture
model of New Zealand white rabbits and the magnesium implants degradation accompanied by uninhibited bone
healing were observed. To determine the relationships between efficacy of the implant device and the bone
healing degree, the histological analysis and X-ray image of the magnesium implants were tested regularly for 4
and 8 weeks.
During the 8 weeks of implantation, no abnormalities were found in the histological examination of the
heart and kidney tissues. The key parameter of the heart and kidney tissues such as the volume and shape of
glomerular were all in a normal level. The serum Mg2+ was lower than the security line (2.0 mol L-1). The Mg2+
concentration in serum of the rabbits is similar to human beings. When the concentration is over 2 mmol L-1,

17
disturbance of magnesium metabolism appears.
The magnesium implants degraded gradually by corrosion in body fluid environment. Equation (8) presents
the chemical reaction of the magnesium degrading process:
Mg+2H2O→Mg2++H2+2OH- (8)
The equation indicates that H2 is produced in the reaction. The production of H 2 may cause gas
accumulation, which is highly concerned in clinical use. The phenomenon of the gas accumulation is commonly
found in the previous studies. For example, Amy Chaya [1] implanted pure magnesium plates and matched screws
into the ulnae of rabbits. Although with the same situation of implantation, 6 gas pockets were found in two
animals of the 12 animals totally in the first 5 weeks. They suspect the rapid corrosion rate caused the gas
accumulation.
However, in the present study, no subcutaneous emphysema was found during the 8 weeks of all the
implantation groups from neither the radiographic results or through visual inspection. The results reflect a large
gap of corrosion rates between pure magnesium and AZ31 alloy in vivo. Sayuri [48] implanted pure magnesium
and AZ31 magnesium alloy in mice subcutaneously and they found that the pure magnesium degradation yielded
a net decrease in volume of 1.4 mm3 compared with the AZ31 magnesium alloy of 0.5 mm3 after 8 weeks.
Furthermore, the implantation site is also an important factor, affecting the corrosion rate. When compared with
the abundant blood supply in the marrow cavity, the body fluid in the muscle tissue or under the subcutaneous is
less, which means the Mg based implants would suffer from a moderate corrosion rate there. Thus, a small
quantity of gas production may be absorbed by the surrounding tissues. Kruas et al. [49] implanted magnesium
pins in the rats. They showed that the generated gas was largely absorbed by the surrounding tissue. Witte F [16]
suggested that the gas was mainly eliminated by the local flood flow. Though H2 released with the reaction of
magnesium degradation and the gas pocket was detected commonly, it is normal that no gas accumulation
produced when in a moderate corrosion situation. In addition, some previous studies [11, 50] revealed that the
released gas did no harm to animals and had no adverse on new bone formation.
4.2 Bone response to magnesium ion
As one formation element of bone, magnesium plays a regulating role in the metabolism of mineral
substance [51]. In the present study, the fracture model was designed to make a 3 mm wide defect in the radius
of the New Zealand white rabbits. Therefore, it can be deduced that the bone callus detected by the X-ray images
over the defected sites was the newly formation bone actually.
To investigate the relationship between new bone formation and implant degradation, different thickness of
MAO coatings were produced on the magnesium implant. As a result, the degradation velocities of different
implants varied.
Compared with the S-F group, the implanted groups all showed healing promoting and the U-Mg group
promoted best followed by CPS10, which indicated that the main factor that promoting bone fracture healing was
the concentration of releasing Mg2+. Moreover, the histological analysis and radiographic evaluation demonstrate
an exact result that Mg2+ ion facilitates fracture healing rate by comparing the degree of fracture healing among
the 4 groups.
The decomposed magnesium, which created OH-, caused local alkaline environment. The localized alkaline
environment and releasing of Mg2+ may induce the protein (BMP-2) secreting by the surrounding tissues, which
could induce the new bone formation, when the concentration of OH - and Mg2+ reached the threshold[18]. This
part of the Mg2+ ion participated in the new bone regeneration but mostly went into the circulatory system and
metabolized. The concentration of Mg2+ ion in the circulatory was tested and the results were showed in Fig. 5.
The MAO coatings fabricated on the Mg implants prevent the alloy from corroding too fast. In the process
of implantation, the coatings degraded gradually, so that the elements of Ca, Si, P and Mg were released in vivo.

18
Silicon is regarded as an essential role in the formation of cross-links between collagen and proteoglycans during
bone growth, preventing the enzymatic degrading and promoting collagen stabilization [52].The promoting
osteoblast proliferation were detected when in the locality containing more silicon element [29]. The Ca and P
elements are helpful to the adsorption of osteoblasts as well [53]. In the present study, the promoting bone
healing of the CPS10 group and CPS20 group were the combined effect of the released Mg element and Ca, Si, P
elements. However, when compared with the bone healing degree of CPS10 and CPS20 groups, the result
revealed a faster healing speed in the CPS10 group according to both the histology tests (Fig. 3) and the X-ray
images (Fig. 4). One possible reason was that the released concentration of Mg2+ in the CPS10 group was higher
than the CPS20 group, since the CPS20 plates showed a better corrosion resistance than the CPS10 plates, which
indicated that the released Mg played a major role in our model than the other elements.
The bone fracture healing promoting by the release of Mg2+ ion make the implant meaningful in fracture
application. Here, we observed the in vivo degradation of the implants and the bone fracture healing response in
8 weeks using the 3 mm bone defect model. The model provided an obvious sequence showing the new bone
formation in the defect site. Therefore, it is simple to understand the forgoing discussion of the promoting healing
by the releasing Mg2+ ion when compare the new bone formation rate among the three implant groups. Several
researchers have found the newly formed bone cell around the magnesium implant through μ-CT methods [37,
54] or histological tissue analysis [30] and they considered the releasing of Mg2+ ion by implant degradation was
the main reason. Zhang [55] et.al discussed the mechanism of new bone formation promoting by Mg2+ ion , as
shown in Fig. 7 (a). They implanted pure magnesium pins into the bone marrow of the femur in rats (not fracture
model) and found abundant new bone formation at peripheral cortical sites. The releasing Mg2+ ion stimulates
the periosteum secreting neuronal calcitonin gene-related polypeptide -α (CGRP), which induces the new bone
formation. Different from Zhang’s implantation model, the implants in our model were located near the
periosteum, thus the releasing Mg2+ ion easily diffuses around the 3 mm bone defect site. Fig. 7 (b) shows a
possible mechanism model of the present study. To determine the effect of Mg2+ in new bone formation
mechanism, we set up a graded releasing concentration of Mg2+ ion, which were adjusted by the controllable
corrosion rate using MAO coatings of 10 μm and 20 μm thickness. As a particular fracture healing case of New
Zealand white rabbits, the bone detect healing period was the main evaluation index, which exhibited a positive
correlation with the releasing Mg2+ concentration. Though the phenomenon of which the Mg2+ ion facilitating
new bone formation has been found, the mechanism especially on the cell scale remain elusive. One possible
mechanism was shown in Fig. 7. When the concentration of Mg2+ ion was over a certain threshold, the ipsilateral
dorsal root ganglia (DRG) was stimulated and secreting CGRP, which induces the relating protein production, such
as CALCRL-and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7
(also known as osterix), resulted in the facilitating of osteogenic differentiation so that the new bone formation
was promoted [55].
Though fast degradation of the implants means better bone fracture healing, but causes a loss of
mechanical integrity and some other over degradation problems such as emphysema, inflammation caused by
alkaline environment. A balance between the bone fracture healing promoting and degradation rate of the
implants should be considered depending on the requirements for different implanting micro environment. In
this case, the MAO coatings demonstrate the great potentials to adjust the corrosion rate by controlling the
coating thickness. Mg degradation and bone fracture healing promoting must be tailored by MAO coating with
different thickness for potential clinic application.

5. Conclusion

19
 In the present study, the uncoated magnesium (U-Mg) plates, 10 m (CPS10) and 20 m (CPS20) thick
microarc oxidation (MAO) coated magnesium plates were implanted into the radii of rabbits using a bone fracture
model of 3mm width. The CPS10 and CPS20 MAO coatings are composed of MgO, Mg2SiO4, CaSiO3 and Mg3(PO4)2
phases. The serum Mg2+ concentration, cardiac histological detection combined with the renal histological
detection results confirmed the biosecurity of the uncoated and MAO coated magnesium implants. The in vivo
studies indicated that the released Mg2+ promoted new bone formation. Both the in vitro electrochemical tests
and in vivo degradation indicated that the corrosion resistance of the magnesium implants was improved by the
MAO coatings. The releasing Mg2+ by the degradation of implants implanted into the fracture site improved the
bone fracture healing, which is attributed to the magnesium promoting CGRP-mediated osteogenic
differentiation. Mg degradation and bone fracture healing promoting can be tailored by microarc oxidation
coating with different thickness for potential clinic application. The conclusion suggests a potential of the MAO
coated magnesium being a temporary load-bearing device application.

Acknowledgement
The partial supports from the NSFC grant nos. 51371071, 51571077 and 51621091, National Basic
Science Research Program (2012CB933900), the Fundamental Research Funds for the Central Universities (HIT.
BRETIII.201202) and the program for New Century Excellent Talents in University of China (NCET-08-0166) are
gratefully acknowledged.

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23
 Magnesium Plate

Radius

Ulna

3 mm wide bone defect

Fig. 1. A bone fracture defect model of 3 mm width for magnesium plate implantation in the radius of New
Zealand white rabbits.

(a1) Mg (b1)
(c) MgO
Mg2SiO4
CaSiO3
Mg3(PO4)2
Intensity A.U.

CPS20

CPS10
50 μm
50 μm
10 20 30 40 50 60 70 80 90
2 θ/degree
-1.0
(a2) (b2)
(d)
-1.2
Coating Substrate Resin
-1.4
Substrate Resin
U (V)

Coating
-1.6

U-Mg
-1.8
CPS10
CPS20
20 μm
-2.0 20 μm
-8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0
-2
log I (A cm )

Fig. 2. SEM morphologies of CPS10 MAO coated magnesium plates (a1, a2) and CPS20 MAO coated
agnesium plates (b1, b2), XRD pattern of CPS10 and CPS20 magnesium plates (c), Polarization curves of U-Mg,
CPS10 and CPS20 plate samples in SBF (d).

24
 U-Mg group CPS10 group CPS20 group

4 Week

8 Week

Fig. 3. Bone tissue detections of the implanted animals after 4 and 8 weeks postoperatively.

25
 U-Mg plate CPS10 plate

8 Week

CPS20 plate S-F plate

U-Mg plate CPS10 plate

4 Week

CPS20 plate S-F plate

Fig. 4. X-Ray Observation of the rabbits after 4 and 8 weeks postoperatively and the taken out implants (The
white arrows pointed at the fracture site and the black arrows pointed at the implants).

26
 U-Mg CPS10 CPS20 S-F

Magnesium ion concentration(mmol L-1)

2

0
4 8
Implantation time (Week)
Fig. 5. Serum Mg2+ concentration results in different implantation time for 4 groups of rabbits after surgery.

27
 (a) U-Mg group CPS10 group CPS20 group

4 Week

8 Week

(b) U-Mg Group CPS10 Group CPS20 Group

4 Week

Fig. 6. Renal histological detections of the implanted animals after 4 and 8 weeks (a), Cardiac histological
detections of the implanted animals after 4 and 8 weeks (b).

8 Week

28
(a) (b)

Fig. 7. The mechanism of bone formation promoting by releasing Mg2+ ion: (a) the model
in Ref. [55]; (b) a 3 mm bone defect model in the present study.

29
 Table 1. Polarization parameters of U-Mg, CPS10 and CPS20 plate samples.

Ucorr (V) Icorr (A cm-2) Rcorr (Ω cm2)

U-Mg -1.778 3.708×10-5 2.848×102
CPS10 -1.532 2.133×10-7 3.145×104
CPS20 -1.391 2.482×10-7 5.160×104

30