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In vitro and in vivo comparison of binary Mg alloys and pure Mg

Article  in  Materials Science and Engineering C · December 2015

DOI: 10.1016/j.msec.2015.12.064

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In vitro and in vivo comparison of binary Mg alloys and pure Mg

Anastasia Myrissa, Nezha Ahmad Agha, Yiyi Lu, Elisabeth Martinelli,

Johannes Eichler, Gabor Szakacs, Claudia Kleinhans, Regine Willumeit-
Römer, Ute Schäfer, Annelie-Martina Weinberg

PII: S0928-4931(15)30679-2
DOI: doi: 10.1016/j.msec.2015.12.064
Reference: MSC 6057

To appear in: Materials Science & Engineering C

Received date: 16 October 2015

Revised date: 15 December 2015
Accepted date: 28 December 2015

Please cite this article as: Anastasia Myrissa, Nezha Ahmad Agha, Yiyi Lu, Elisabeth
Martinelli, Johannes Eichler, Gabor Szakacs, Claudia Kleinhans, Regine Willumeit-
Römer, Ute Schäfer, Annelie-Martina Weinberg, In vitro and in vivo comparison of
binary Mg alloys and pure Mg, Materials Science & Engineering C (2015), doi:
10.1016/j.msec.2015.12.064

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In vitro and in vivo comparison of binary Mg alloys and pure Mg

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Anastasia Myrissa1, Nezha Ahmad Agha2, Yiyi Lu2, Elisabeth Martinelli1, Johannes Eichler1,

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Gabor Szakacs2, Claudia Kleinhans1*, Regine Willumeit-Römer2, Ute Schäfer3, Annelie-

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Martina Weinberg1

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Department of Orthopedics and Orthopedic Surgery, Medical University Graz, 8036 Graz,

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Austria
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Institute of Materials Research, Division Metallic Biomaterials, Helmholtz-Zentrum
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Geesthacht (HZG), 21502 Geesthacht, Germany
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Department of Experimental Neurotraumatology, Medical University Graz, 8036 Graz,
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Austria
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* Corresponding Author; Phone +436607177929; Claudia.Kleinhans@medunigraz.at

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Abbreviation

DR Degradation Rate

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DMEM Dulbecco's Modified Eagle's Medium

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µCT micro Computed Tomography

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BN Boron-nitride

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SEM Scanning Electron Microscopy

BSE Back Scattered Electron

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EDX Energy Dispersive X ray

FBS Fetal Bovine Serum

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PBS Phosphate Buffered Saline
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Abstract

Biodegradable materials are under investigation due to their promising properties for

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biomedical applications as implant material. In the present study, two binary Magnesium

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(Mg) alloys (Mg2Ag and Mg10Gd) and pure Mg (99.99%) were used in order to compare the

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degradation performance of the materials in in vitro to in vivo conditions.

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In vitro analysis of cell distribution and viability was performed on discs of pure Mg, Mg2Ag

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and Mg10Gd. The results verified viable pre-osteoblasts cells on all three alloys and no

obvious toxic effect within the first two weeks.

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The degradation rates in in vitro and in vivo conditions (Sprague–Dawley® rats) showed that

the degradation rates differ especially in the 1st week of the experiments. While in vitro
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Mg2Ag displayed the fastest degradation rate, in vivo, Mg10Gd revealed the highest
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degradation rate. After four weeks of in vitro immersion tests, the degradation rate of Mg2Ag
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was significantly reduced and approached the values of pure Mg and Mg10Gd. Interestingly,
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after 4 weeks the estimated in vitro degradation rates approximate in vivo values.
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Our systematic experiment indicates that a correlation between in vitro and in vivo

observations still has some limitations that have to be considered in order to perform

representative in vitro experiments that display the in vivo situation.

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Keywords: Magnesium alloys, biodegradable implants, degradation rate, in vitro, in vivo

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1. INTRODUCTION

Bioresorbable materials are under investigation because of their promising properties for

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biomedical applications such as vascular stents or orthopedic implants. As orthopedic

devices, biodegradable Magnesium (Mg) implants can be used in patients with bone

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fractures and consequently due to their degradation ability no second surgery for implant

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removal is necessary [1, 2]. Biodegradable Mg exhibits high biocompatibility and low stress

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shielding due to the elastic modulus which is similar compared to bone tissue [2, 3].

However, Mg implants can display uncontrolled and high degradation rates that need to be
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considered in order to be used as medical device [4, 5]. As a consequence of too fast

degradation, high amounts of hydrogen gas are released, and the pH and the ion
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concentrations of Mg and alloying elements increase in the surrounding tissue. These

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unwanted chemical effects can be diminished in several ways by tailoring either the

microstructure or the surface properties [6].

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The microstructure and thus the degradation rate can be tailored by alloying and / or heat

treatment or using wrought materials. Suitable chemical elements such as Al, Zn, Mn, Ca, Y,
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Zr and Rare Earth Elements (REE) are used to enhance the mechanical and physical

properties of Mg alloys [7]. Various Mg alloys have been investigated in in vitro or in in vivo

conditions, however only few were performed on the same material. Even less have been

tested in both, in in vitro and in vivo conditions to compare their degradation behavior [8]. On

the one hand, degradation profiles were studied in in vitro conditions using immersion tests

[9]. Furthermore, cytotoxicity and biocompatibility were examined using primary cells [10] or

different cell line cultures [11, 12]. On the other hand, the degradation profile in in vivo

conditions was studied using the non-destructive method of micro computed tomography

(µCT) and analyzing the material volume during bone healing process [1, 6, 13].

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In many cases, the alloys’ compositions contain more than one alloying element in order to

obtain more flexibility in terms of processing parameters, degradation and mechanical

properties [14, 15]. In other cases, Mg binary alloys are used in order to discover the effect of

only one element in the biological in vitro system and these results can be used as guidance

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for future selection of the alloying elements to produce Mg alloys [12]. Within this study, we

chose binary alloys to reduce the influence of various alloying elements on the degradation

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performance of the implant and on cell interaction. As a result, possible changes in

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degradation behavior are caused by one single alloying element.

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The chemical elements used in our study were Gadolinium (Gd) and Silver (Ag). Mg alloys

containing REEs have shown increased mechanical properties and corrosion resistance of
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the material [16-18]. It is known that an increased amount of Gd (up to 10%) within the

material improves the corrosion performance. However, Gd contents higher than 10 % do not
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further enhance corrosion resistance [17]. From the biological point of view, it is mentioned
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that REEs are used in anticancer drugs because of their anti-carcinogenic properties [19,

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Ag addition to Mg also improves the mechanical properties and increases the corrosion

resistance of these materials when a suitable heat treatment is applied. Moreover, it was
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reported that Mg-Ag alloys show good biocompatibility and satisfactory antibacterial

properties [11].

In this study, two binary Mg alloys and pure Mg were used to compare the degradation

performance of the materials in in vitro and in vivo conditions. The in vitro degradation rate

was calculated through the weight loss method. Pin volume and surface data from in vivo

µCT were used to calculate the in vivo degradation rate. In addition, the microstructure of the

materials as well as their cytotoxicity was analyzed in order to compare the different Mg

alloys regarding their performance.

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2. MATERIALS AND METHODS

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2.1 Materials production and sterilization

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Three Mg-based materials were used in this study: Mg2Ag, Mg10Gd, and pure Mg. The

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materials were cast at Helmholtz-Zentrum (HZG), Geesthacht, Germany. For the casting,

pure materials were used: Mg (99.99%), Gd (99.95%), Ag (99.99%).

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The Mg2Ag was produced by permanent mould gravity casting. After melting the pure Mg,
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the melt was hold at 720 °C and the preheated Ag was added with continuous stirring for 15

minutes (min). The melt was poured into a preheated (550 °C) permanent steel mould using
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Boron-nitride (BN) as mould release agent.

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Mg10Gd and pure Mg were produced by permanent mould direct chill casting. In case of this
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casting method the melt was poured into preheated steel mould. The mould with the melt
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was placed into a holding furnace at 680 °C for further 15 min. After the holding time, the

mould was slowly immersed into running water in order to solidify the ingot. Again BN was
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used as mould release agent. During the casting processes, the same protective atmosphere

with 2 wt % SF6 in Ar was used. The alloys were homogenized with a T4 heat treatment prior

to extrusion in Argon (Ar) atmosphere at 550 °C (Mg10Gd) and at 420 °C (Mg2Ag) for 6

hours (h). The homogenized ingots were hot extruded indirectly. The extrusion parameters

are presented in Tables 1 and 2. Different processing parameters in casting and extrusion

have been applied due to the diversity of the materials and to achieve similar microstructures

in the final products.

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Table 1: Extrusion parameters for rods used to prepare discs to be used in in vitro cell

experiments.

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Extrusion parameters
Alloy extrusion Extr. T dfinal

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Type Speed [mm/s] Billet T [°C]
ratio [°C] [mm]

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Mg Indirect 1:84 0.7 300 340 12
Mg2Ag Indirect 4:25 2.5 370 370 12

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Mg10Gd Indirect 4:25 MA 3.5 370 430 12

Table 2: Extrusion parameters for rods used to prepare pins for in vivo implantation.

Extrusion parameters
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Alloy extrusion
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Type ratio Speed [mm/s] Extr. T [°C] Billet T [°C] dfinal [mm]
Mg Indirect 1:67 0.7 300 340 6
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Mg2Ag Indirect 1:19 2.5 370 400 3

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Mg10Gd Indirect 1:67 0.7 300 400 6

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For in vitro cell experiments, discs (Ø=10 mm, h=1.5 mm) were produced from the extruded

rods according to defined extrusion parameters (Table 1). The pins (Ø=1.6 mm, h=10 mm)

used in the in vitro and in vivo tests were also produced from extruded rods and the extrusion

parameters are shown in table 2. The extruded pure Mg and Mg10Gd rods (d=6 mm) were

reduced to 1.6 mm by turning (Ernst Wittner GesmbH, Vienna, Austria). The extruded Mg2Ag

rods were drawn to wires using the hardened steel drawplates with different wire gauge

combined with drawing bench in order to reduce the thickness of wire by reshaping the

metal. The wires were annealed at 300 °C for around 45 min in the furnace between 1-3 wire

drawing steps. The wires of Mg2Ag were drawn to 1.6 mm of diameter. After production, the

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samples were sterilized by gamma radiation dose at 29.2 KGy (performed by the company

BBF GmbH, Stuttgart, Germany).

2.1.1 Chemical composition and density

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The contents of Mg, Fe, Cu and Ni were determined by spark emission spectrometer

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(Spectrolab M, Spektro, Germany) and the contents of Ag and Gd were determined by X-ray

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fluorescence spectrometer (Bruker AXS S4 Explorer, Bruker AXS GmbH., Germany). The

density of the pins (Ø=1.6 mm) was determined in ethanol by Archimedean principle.

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Table 3: Chemical composition and density of pure Mg, Mg2Ag and Mg10Gd pins were
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analyzed by using a spark emission spectrometer and the Archimedean principle

respectively.
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Chemical composition wt.% Density ρ

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Alloy
Ag Gd Fe Cu Ni Mg [g/cm³]
Pure Mg - - 0.0052 0.0023 0.0014 Bal. 1.740
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Mg2Ag 1.75 - 0.002 0.0019 0.0013 Bal. 1.748

Mg10Gd - 10.5 0.0029 0.0048 <0.0036 Bal. 1.876
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2.1.1 Microstructure analysis with optical- and scanning electron

microscopy

The specimens were embedded in plastic cold-curing resin. Powdered and liquid Demotec

30 was mixed in the ratio 1:1. The plastic resin took approximately 15 min to be solidified

under room temperature. Specimens were ground using silicon carbide emery paper up to

2500 grit, and then polished with a lubricant containing 1μm diamond particles and 0.05 µm

colloidal silica (OPS). The polished surface was finally cleaned using ethanol and dried under

blowing hot air. Specimens were etched in an etchant containing 30 ml deionized water, 140

ml ethanol, 7 ml glacial acetic acid and 8 g picric acid, and quickly washed using ethanol and
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dried with blowing hot air. Microstructures were observed using optical microscope (Reichert-

Jung MeF3) with a digital camera. The grain size was determined using the line intercept

method [21]. Scanning Electron Microscopy (SEM) was performed on the as-solidified

samples with a VEGA3 Tescan microscope, operating at 15 kV using back scattered electron

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(BSE) imaging mode. The SEM is equipped EDAX energy dispersive X ray (EDX)

spectrometer operated at 15 kV.

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2.2 In vitro methods
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2.2.1 In vitro degradation rate by mass loss method
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Before sample sterilization, the initial weight of the samples was recorded. 5 samples were
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immersed in 3 ml / sample of Dulbecco's modified eagle's medium (DMEM) supplemented

with 10% fetal bovine serum (FBS) for 168 h (1 week) under cell culture conditions (37 °C,
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20% O2, 5% CO2, 95% rH). The same immersion set up test was conducted with pin samples

for 4 weeks, too. Samples lay on surface during the immersion time. The immersion medium
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was changed every 2-3 days to present a semi-static immersion test and to avoid saturation

effects. After immersion, the subsequently formed products were removed by treating the

corroded disk with chromic acid (180 g/L in distilled water, VWR International, Darmstadt,

Germany) for 20 min at room temperature. Then the degradation rate was calculated in

mm/year using the equation:

DR = 8.76 . 104 . Δg / A ∙ t ∙ ρ ,

where Δg is the weight change in grams, A is the surface area of the sample in cm 2, t is the

immersion time in hours, and ρ is the density in g/cm3.

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Pictures of pure Mg, Mg2Ag and Mg10Gd pins after immersion test and after chromic acid

treatment were taken by Moticam10 (Motic Deutschland GmbH, Wetzlar, Germany).

Samples after 4 weeks of immersion, and after the chromic acid treatment, were analyzed by

Phenom ProX desktop SEM machine (Phenom-World BV, Eindhoven, Netherlands).

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2.2.2 In vitro viability by LIVE/DEAD (viability/cytotoxicity) staining

assay

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In vitro qualitative analysis of cell coverage and viability was performed by using LIVE/DEAD
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(Life technologies, Darmstadt, Germany) assay. Primary pre-osteoblasts were used to

perform the test and were isolated and characterized according to Burmester et al. [22]. The
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isolation of cells was approved by the local ethics committee and performed according to the
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Declaration of Helsinki as described in [23]. In short description, cancellous bone material

was removed from the hip joint and transferred to a cell culture flask. Then the cancellous
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particles were incubated in cell culture medium until the cell growth confluence became 90%
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and then they were divided. The used osteoblasts were below the third passage. The test

protocol started by pre-incubating the discs (Ø=10 mm, h=1.5 mm) for 24 h, then 105 primary
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osteoblasts were seeded on each disc surface for 13 days (n=3 disc samples per Mg alloy).

The chemical composition of the discs was analyzed previously and revealed the same

composition as the pins. Subsequently, the staining solution was prepared by adding 4 µl

Calcein AM (LIVE) and 10 µl Ethidium homodimer-1 (DEAD) to 10 ml of Phosphate Buffered

Saline (PBS). The samples were first washed with PBS to eliminate non-adherent cells,

followed by immersing each sample with 1.5 ml of staining solution and incubate it under a

5% CO2 atmosphere at 37° C for 20 min. Then the staining solution was replaced by DMEM

and samples were visualized by fluorescent microscope (Nikon GmbH, Düsseldorf,

Germany), the applied filters were FITC (Ex: 460-500 nm; Em: 510-560 nm; Mirror at 505

nm) and Texas red (Ex: 540-580nm; Em: 600-660; Mirror at 595 nm).
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2.3 In vivo methods

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All the experiments on animals were conducted under ethical respect for animals and were

authorized by the Austrian Ministry of Science and Research (accreditation number BMWF-

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66.010/0078-II/3b/2011). 18 male Sprague–Dawley® rats (n=6 rats/per group) of 140-160

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grams weight and 5 weeks of age were used for this study. Cylindrical pins with 1.6 mm

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diameter and 8 mm length of pure Mg (99.99%), Mg2Ag and Mg10Gd were used for the

implantation. The implantation surgery took place under general anesthesia and two identical
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pins were implanted transcortically in the femoral bones of each rat. The whole surgical

procedure and the postoperative treatment are described by [1]. µCT scans were performed

at 3 different time points, 1 week, 4 weeks and 12 weeks after the operation. During the µCT
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scans, the animals were anesthetized with volatile isoflurane (Forane#, Abbot AG, Baar,

Switzerland). The µCT scan software used was the Siemens Inveon Acquisition Workplace
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1.2.2.2. Scans of the rats were performed at 70 kV voltage, 500 µA current, and 1000 ms
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exposure time. 3-D morphometric analysis and the pin volume and surface were evaluated

by using the software Mimics (Version 15.0 and 17.0, Materialise, Leuven, Belgium) as
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previously described by Kraus et al [1]. Degradation rate (n=6 bones per each time point per

group) was calculated by the equation:

DRi = Δxi / Δt with Δxi = ΔVi / Si,

where i is the observation time point, ΔVi is the change of the volume between the

observation time point i-1 and I (Δt) in mm3 and Si is the surface area at the observation time

point I in mm2 [6].

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2.4 Statistical analysis

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The results were analyzed by the SPSS statistical analysis software. The in vitro degradation

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rates of the pins were analyzed by ANOVA one way test. The significance level was set up

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on the p value p<0.05, p<0.01 and p<0.001. The results of the in vivo degradation rates were

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analyzed with SPSS Mann Whitney-U-test. The significance level was set up on the p value

of 0.05.

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3. RESULTS

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To evaluate the degradation performance of two binary Mg alloys and pure Mg in in vitro and

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in vivo conditions, the weight loss method was used to calculate the in vitro degradation rate.

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The in vivo degradation rate was calculated by the volume and surface data of pins

implanted in the femoral bone of Sprague–Dawley® rats. In addition, the microstructure was

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characterized to determine its influence on the degradation behavior.
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3.1 Optical microstructure

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Pins of pure Mg, Mg2Ag and Mg10Gd with a diameter of 1.6 mm and length of 8 mm were
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produced and analyzed concerning grain size, optical microstructure and grain distribution
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(Figure 1).
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The optical microstructure of all materials showed similar grain size distrubution in transverse

direction, which indicated homogeneous material and complete recrystallization after

extrusion. Mg10Gd displayed the largest average grain sizes with 25.8 ± 14.2 µm, pure Mg

21.0 ± 13.1 µm and Mg2Ag an average grain size of 18.19 ± 11.98 µm. Especially in

longitudinal direction the influence of the extrusion was visible (Figure 1B). In Mg2Ag and

Mg10Gd the grain size was also small but more homogeneously distributed compared to

pure Mg (Figure 1A and1B).

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Figure 1: (A) Estimation of the average grain sizes of pure Mg, Mg2Ag and Mg10Gd pins

with diameter of 1.6 mm by using the line intercept method. (B) The optical microstructure of

pins in transverse direction and in longitudinal direction was detected by optical microscope

with a digital camera.

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In the case of Mg10Gd specimes, small particles are observed alongside the extrusion

direction in the optical microscope images (Figure 2). On the SEM image (Figure 2A), these

brighter particles are the most possible intermetallic phase Mg5Gd from the phase diagram

[24] and/or GdH2 [25]. The EDX point analysis from a certain rectangular particle and from

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the matrix are presented in Figure 2B. These particles are mainly containing Gd, thus they

are more probably GdH2 particles. For pure Mg and Mg2Ag similar particles were not found.

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Figure 2: Mg10Gd pin in A) SEM picture, point 1 is certain rectangular particle and point 2 is

from the matrix which are used for EDX analysis and B) EDX analysis of Mg10Gd pins.
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3.2 In vitro degradation rate by mass loss method

All materials were tested for their degradation behavior in DMEM for 1 and 4 weeks by

calculating the mass loss presented in Figure 3. The estimated degradation rates in mm/year

within the 1st week revealed the highest degradation rate for Mg2Ag with 1.81 ± 0.19

mm/year whereas pure Mg exhibited a rate of 0.75 ± 0.45 mm/year and Mg10Gd 0.56 ± 0.04

mm/year. 4 weeks after immersion, the degradation of Mg2Ag (0.36 ± 0.01 mm/year) had

been significantly reduced. The degradation rate of pure Mg (0.32 ± 0.04 mm/year) is

reduced, but not significantly, and the degradation rate of Mg10Gd (0.56 ± 0.39 mm/year) did

not show any difference. After 4 weeks of immersion, no significant differences on the

degradation rates can be seen between these three materials.

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These calculated degradation rates correlated with macroscopically images of the pins after

immersion for 1 week, displayed in Figure 4A. Pins made of Mg10Gd showed only few

changes in surface morphology compared to the pitting corrosion detected on Mg2Ag and

partly on pure Mg pins. In Figure 4B, macroscopic images of the pins after immersion for 4

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weeks showed morphological changes of the surface. The SEM images, in high

magnification, of pure Mg (Figure 4B-d and g) pins revealed the formation of pits on the

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surface. SEM images of Mg2Ag (Figure 4B-e and h) revealed localized corrosion morphology

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around the defects in the metallic bulk. SEM images of Mg10Gd (Figure 4B-f and i) showed

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its localized corrosion. MA
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Figure 3: Calculated degradation rates [mm/year] of pure Mg, Mg2Ag and Mg10Gd pins by

in vitro immersion tests (n=5/Mg alloy) 1 and 4 weeks after the immersion. The results are

expressed as the average ± standard deviation of the degradation rate [mm/year] to the

different time points [weeks]. *, ** and *** indicate the significant difference of value p<0.05,

0.01 and 0.001, respectively. The statistical analysis was performed with the ANOVA one

way Test of SPSS statistical analysis software.

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Figure 4: Macroscopic images of pure Mg, Mg2Ag and Mg10Gd pins revealing surface

morphology after the in vitro immersion test for 1 week (A) and for 4 weeks (B - a, b and c).

SEM images of pure Mg (d and g), Mg2Ag (e and h) and Mg10Gd (f and i) showing the

morphological changes of the pins surface after 4 weeks in immersion media.

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3.3 In vitro viability by LIVE/DEAD (viability/cytotoxicity) staining assay

In vitro qualitative analysis of cell distribution and viability was performed on pure Mg, Mg2Ag

and Mg10Gd by LIVE/DEAD staining after 13 days of culture in proliferative medium. Primary

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pre-osteoblasts were seeded on the surface of the discs. Figure 5 shows a homogenous cell

distribution throughout the surface. Viable cells are marked in green and dead cells in red.

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The results revealed viable cells on all alloys.

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Figure 5: Fluorescent microscope images in 20x magnification after LIVE/DEAD staining of

primary pre-osteoblasts cultured on Mg samples for 13 days, where viable cells are labeled

in green and dead cells in red (n=3 disc samples per Mg alloy). Tissue culture polystyrene

was used as control.

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3.4 µCT

The µCT images in Figure 6 show the degradation of pure Mg, Mg2Ag and Mg10Gd at 1, 4

and 12 weeks post-operative.

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Figure 6: µCT reconstructions (two-dimensional slices) showing the degradation process at

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1, 4 and 12 weeks after the implantation in the femur bone of Sprague–Dawley® rats (n=6
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rats/per group).

The µCT images of pure Mg implanted bones revealed gas bubbles in the intramedullary
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cavity in the 1st week after the implantation, whereas 4 weeks after the operation and after

12 weeks no gas formation could be seen. New bone formation around the pin was clearly

obvious 12 weeks after the operation, however, no degradation of the implant occurred.

Mg2Ag produced high amount of gas in the intramedullary cavity and also in the surrounding

tissue 1 week post-operative. After 4 weeks, gas decreased clearly and was noticeable only

in the medullary cavity around the implant. After 12 weeks, the gas formation seemed to be

further reduced and new bone formation was obvious. The implant did not show any

degradation.

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In the µCT images of the Mg10Gd implanted bones, low amount of gas was obvious the first

week post-operative. 4 weeks after implantation increased gas cavities were exhibited

especially in the surrounding tissue and degradation is visible. After 12 weeks no obvious

gas cavities were visible. In addition, bone contact to the surface of the implants succeeded

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since the 4th week. At 12 weeks post operation, pins were broken and completely reduced to

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small pieces surrounded of new formed bone.

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3.5 Degradation rate in in vivo conditions

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Figure 7: Degradation rates of pure Mg, Mg2Ag and Mg10Gd in mm per year versus time

(n=6 bones/Mg alloy). The degradation rate of Mg10Gd cannot be calculated 12 weeks after

the implantation due to the degradation performance. The results are expressed as the

average ± standard deviation of the degradation rate [mm/year] to the different time points

[weeks]. * indicates the significant difference in terms of p<0.05. The statistical analysis was

performed with the Mann Whitney-U-Test of SPSS statistical analysis software.

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The degradation rate of pure Mg started with 0.4 mm/year but decreased to less than 0.2

mm/year at 4 and 12 weeks after the operation. The degradation velocity of Mg2Ag began

with more than 0.2 mm/year, at 4 weeks increased moderately, but not significantly, and at

12 weeks reached the same level as the DR of pure Mg. The degradation velocity of

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Mg10Gd was more than 0.6 mm/year at first week’s time point and reduced to almost 0.5

mm/year 4 weeks after the implantation. The pin volume and surface and consequently the

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degradation rate could not be calculated 12 weeks after the implantation due to the

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degradation performance. The Mg10Gd implant degradation was not significantly higher than

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the one of pure Mg and Mg2Ag implants in the first week but it was significantly higher than

pure Mg and Mg2Ag 4 weeks after the operation.

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Figure 8: Comparison of the degradation rates of each magnesium alloy at 1st and 4th week

in in vitro immersion test to in vivo experiments.

Comparing the in vivo and in vitro results, we observed different degradation performances

especially at the first week of our experiments. In Figure 8, the degradation rate of Mg2Ag

revealed, after one week in immersion test, the highest degradation rate of all materials in in
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vitro and in vivo conditions. After 4 weeks, the degradation rate of Mg2Ag decreased to the

same level as the other 2 materials both in in vitro and in vivo conditions. Pure Mg pins

showed two times higher in vitro degradation compared to the in vivo situation at 1st week.

However, at 4 weeks’ time point the degradation slowed down. The degradation rate in case

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of Mg10Gd is equal in in vitro and in vivo conditions both at 1st and 4th week. Moreover, after

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4 weeks there are no significant differences on the degradation rates between the materials.

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4. DISCUSSION

As degradation velocity is still a major concern in the application of Mg implants, several

strategies are approached to decelerate the degradation rate [6]. One approach is alloying

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with elements to control the implant degradation rate and therefore to guarantee fracture

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stability for the required time of bone healing. As Ag has promising anti-microbial properties,

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it was selected to investigate the influence on the degradation behavior. Another approach

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was the usage of Gd, which belongs to Rare Earth Elements, known to increase the

mechanical properties and also the corrosion resistance of Mg alloys [17].

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Primarily before implantation into rats, in vitro cell tests were performed to evaluate cell
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viability and the ability to adhere to the analyzed materials in order to exclude a toxic

potential of our selected materials and to guarantee ingrowth of the implants into bone tissue.
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Our LIVE/DEAD staining results indicate that the cells are predominantly healthy and spread
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throughout the surface area. Also the study of Di Tie et al. [11] present a survival rate higher

than 95% of human osteoblasts on pure Mg and Mg2Ag discs respectively, after a culture
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period of 72 h.
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In several studies, the in vitro degradation rate is calculated by the weight loss method [13,
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17, 26-28] or by the gas evolution method [12, 17, 27, 29]. Furthermore, in vitro studies were

performed which determine the average degradation rate [11, 13] or the degradation rate at

different time points [30, 31]. In our study, we calculated the in vitro degradation rate by

weight loss method at two different time points (1 and 4 weeks) in order to investigate the

tendency of the degradation and whether the longer immersion time can affect the

degradation behavior. In our experimental set up, the pin–shaped materials were immersed

in high-glucose DMEM + 10% FBS solution media in a semi-static way, showing that Mg2Ag

featured the highest degradation rate, followed by pure Mg. Mg10Gd displayed the slowest

degradation rate one week after the immersion. The comparison of in vitro degradation rates

at 1st and 4th week (Figure 3) revealed a significantly reduced degradation rate of Mg2Ag

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after 4 weeks. It is well known that the initial degradation rate is higher and slows down over

time [26] due to the formation of a protective layer [32]. This could be the reason why the

degradation rate especially for Mg2Ag is reduced at 4 weeks compared to one week in in

vitro conditions. The degradation rate of pure Mg is also reduced, but not significantly, 4

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weeks after immersion. This result is similar to Hong et al. [31], who showed in their in vitro

set up that the degradation rate of pure Mg decreases over time due to the formation of a

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protective layer on the surface of the implants. On the contrary, in case of Mg10Gd pins, the

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protective layer could have been already formed within the first week as we could not

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observe any difference on the degradation rate between the 1st and 4th week of our in vitro

immersion test.
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Comparing pure Mg, Mg2Ag and Mg10Gd pins in in vivo conditions, we also observed

different degradation performance at 1 week, 4 weeks and 12 weeks’ time points (Figure 7).
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Mg10Gd showed higher degradation rates in in vivo conditions in comparison to pure Mg and
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Mg2Ag at 1 and 4 weeks after the operation. At 12 weeks post implantation the calculation of

the degradation rate through the pin volume and pin surface was not feasible due to integrity
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loss and disintegration into small remnants of the material.

This difference in the degradation rate of Mg10Gd pins could be explained by the
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intermediate particle distribution. The microstructure of Mg10Gd pins showed Gd rich

particles in the specimens (Figure 2). The Gd rich particles GdH2 and Mg5Gd are more noble

than the Mg matrix itself and cause a galvanic couple effect between the matrix and the

particles [33], resulting in a localized corrosion which may lead to the fast degradation and

breakage of the pins. This could be a reason why the in vivo degradation performance of

Mg10Gd is not homogeneous. On the contrary, the formation of Mg4Ag precipitates during

the hot extrusion could be avoided and no sign of this phase could be found on Mg2Ag pins.

This could be one reason why the Mg2Ag showed slower degradation performance in in vivo

conditions.

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The degradation behavior of the Mg alloys is reported to depend on the microstructure of the

materials and their grain size [29, 34], affected also by the material production process itself

[7]. In our study, after extrusion, the diameters of the pure Mg and Mg10Gd rods were

reduced (from 6 mm to 1.6 mm) by turning. In comparison, Mg2Ag rods were drawn to pins

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of 1.6 mm from 3 mm. In general, the grain size of the Mg2Ag pins is smaller, but not

significantly, and more homogeneous in comparison to pure Mg and Mg10Gd. This could

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explain why Mg2Ag pins exhibit smoother and more homogeneous degradation performance

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in our animal short-term study of 12 weeks. Differences on the diameter reduction processes

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may influence the microstructure of the pins and consequently on the degradation behavior

of these materials.
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Martinez Sanchez et al. [8] concluded in a recent review that in vivo degradation occurs

slower than estimated from in vitro experiments. Comparing our in vivo and in vitro results,
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we also see a tendency of higher in vitro degradation rates than in vivo. However, the in vitro
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values are comparable at all time points (Figure 8) except of the Mg2Ag degradation rate at

the first week. Charyeva et al. performed in vitro studies with disc-shaped samples were
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immersed in low glucose DMEM + 10% FBS solution media in a semi-static way, showing a

slower degradation rate for Mg2Ag than for Mg10Gd [35]. Comparing their results to our in
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vivo data, the corrosion tendency is consistent and correlate to our results as the determined

degradation rate in vivo was higher for Mg10Gd than for Mg2Ag. We can hypothesize that

the different DMEM composition regarding the amino-acids affects the degradation

performance of the same materials. Furthermore, it is reported that the ratio of volume

solution to surface area of the sample (V/S) can also affect the degradation rate of the

materials in in vitro immersion tests and the selection of this ratio is very important in case of

in vitro comparison to in vivo degradation rates in each specific implantation model [26]. The

V/S ratio in our in vitro experimental set up is calculated as 5.53 ml / cm2. On the contrary,

the V/S ratio in the experimental set up of Charyeva et al. [35] is calculated to 1.59 ml / cm2,

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showing that the second set up approaches more suitable the in vivo transcortical

implantation animal model.

This effect of a slower material degradation when it is incorporated in the body has been

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observed in many studies [8, 27, 36-39]. Simplified in vitro testing system can hardly mimic

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the complex in vivo situation in a living organism [26]. The degradation rate is influenced by

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many factors such as pH of body fluids, variations in the pH affected by degradation

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products, ion concentration, presence of proteins and the influence of cells in the surrounding

tissues [19, 40]. To approach in vivo conditions, different cell culture media was used in

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several in vitro studies [9, 26, 28, 41]. In our experiments cell culture medium DMEM

supplemented with 10% FBS was selected as corrosion media in order to simulate a more
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realistic corrosion environment by including proteins and ions. The chloride and especially

the bicarbonate ions concentration differ between the blood plasma and DMEM solution. This
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can influence the degradation behaviour by enhancing the MgCO3 formation as a main
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degradation product [42, 43]. The constant blood flow in the animal study is a non-static

system which differs from our semi-static in vitro system. The pH changes in in vivo
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conditions and is regulated by the blood buffer system during the degradation process. In

contrast, the pH in in vitro conditions can be controlled and adjusted. Proteins’ interaction
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and adherence of different cell types play also a key role in the in vivo degradation

performance of the materials [44, 45]. This can explain in some extent the lower values in

vivo and reveal the importance of both proteins and different cells interaction with/nearby the

implant [46]. Moreover, the lack of mechanical exposure in in vitro conditions may cause the

different behaviour of the material accompanied by different local environments.

Taking all of these into consideration, the higher degradation rate in vitro could be related to

the insufficient simulation of a realistic in vivo environment. The in vitro experimental set-up

influences the material behavior and can be tailored by reconsidering immersion parameters

(e.g. V/S, media selection, mechanical stimulation, cell exposition, dynamical set-up).

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5. CONCLUSION

We can conclude out of our experiments, comparing the in vitro and in vivo performance of

two different binary Mg alloys and pure Mg, that in some extend the in vivo degradation rate

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can be displayed in vitro. Mainly in the first week of in vitro immersion, we observed

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differences in the degradation rate that can be due to a protective layer that is formed in

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dependency on the material composition, immersion media or local environment. However,

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comparing the degradation performance after 4 weeks in in vitro and in vivo there is no

significant difference in degradation velocity. Further studies have to been done in order to

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uncover the effect of different immersion solution media on the degradation rates.

Furthermore, studies concentrating on the formation of a degradation layer and the

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coherence with corrosion are necessary. In case of Mg10Gd, we observed the distinct

influence of the alloying element on the material performance. By the selection of e.g. more
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noble elements, the degradation behavior is accelerated by triggering the galvanic couple
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effect and could be used to specifically tailor material characteristics. Future studies should

address the current limitations in in vitro testing systems by applying more complex test set
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ups, including biological components (e.g. relevant cells) and mechanical and dynamical
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exposure of the material in order to predict the in vivo performance.

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Acknowledgements

This work is supported by European FP7 Marie Curie Program (Project Number:289163) and

by Helmholtz Virtual Institute VH-VI-523 (In vivo studies of biodegradable magnesium based

implant materials). The authors appreciate the Institute of Biomedical Research at the

Medical University of Graz for the provision of infrastructure facilities to perform the animal

studies. The authors appreciate the support from Prof. Norbert Hort and Dr. Frank

Feyerabend.

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REFERENCES

[1] T. Kraus, S.F. Fischerauer, A.C. Hänzi, P.J. Uggowitzer, J.F. Löffler, A.M. Weinberg,
Magnesium alloys for temporary implants in osteosynthesis: In vivo studies of their
degradation and interaction with bone, Acta biomaterialia 8 (2012) 1230–1238.

T
IP
[2] C. Castellani, R.A. Lindtner, P. Hausbrandt, E. Tschegg, S.E. Stanzl-Tschegg, G. Zanoni,
S. Beck, A.M. Weinberg, Bone–implant interface strength and osseointegration:
Biodegradable magnesium alloy versus standard titanium control, Acta Biomaterialia 7

R
(2011) 432-440.

SC
[3] H. S. Brar M.O. Platt, M. Sarntinoranont, P. I. Martin, M. V. Manuel. Magnesium as a
Biodegradable and Bioabsorbable Material for Medical Implants, JOM 61 (2009)31-34.

NU
[4] G. Song, Control of biodegradation of biocompatable magnesium alloys, Corrosion
Science 49 (2007) 1696-1701.

[5] M.P. Staiger, A.M. Pietak, J. Huadmai, G. Dias, Magnesium and its alloys as orthopedic
MA
biomaterials: A review, Biomaterials 27 (2006) 1728-1734.

[6] S.F. Fischerauer, T. Kraus, X. Wu, S. Tangl, E. Sorantin, A.C. Hanzi, J.F. Loffler, P.J.
Uggowitzer, A.M. Weinberg, In vivo degradation performance of micro-arc-oxidized
magnesium implants: A micro-CT study in rats, Acta Biomaterialia 9 (2013) 5411-5420.
D
TE

[7] F. Witte, N. Hort, C. Vogt, S. Cohen, K.U. Kainer, R. Willumeit, F. Feyerabend,

Degradable biomaterials based on magnesium corrosion, Current Opinion in Solid State and
Materials Science 12 (2008) 63-72.
P

[8] A.H. Martinez Sanchez, B.J. Luthringer, F. Feyerabend, R. Willumeit, Mg and Mg alloys:
CE

How comparable are in vitro and in vivo corrosion rates? A review, Acta Biomaterialia 13
(2015) 16-31.

[9] J. Hofstetter, E. Martinelli, A.M. Weinberg, M. Becker, B. Mingler, P.J. Uggowitzer, J.F.
AC

Löffler, Assessing the degradation performance of ultrahigh-purity magnesium in vitro and in

vivo, Corrosion Science 91 (2015) 29-36.

[10] K. Pichler, S. Fischerauer, P. Ferlic, E. Martinelli, H.-P. Brezinsek, P.J. Uggowitzer, J.F.
Löffler, A.-M. Weinberg, Immunological Response to biodegradable Magnesium Implants,
Jom 66 (2014) 573-579.

[11] D. Tie, F. Feyerabend, W.-D. Müller, R. Schade, K. Liefeith, K.U. Kainer, R. Willumeit,
Antibacterial Biodegradable Mg-Ag alloys, European Cells and Materials 25 (2013) 284-298.

[12] X. Gu, Y. Zheng, Y. Cheng, S. Zhong, T. Xi, In vitro corrosion and biocompatibility of
binary magnesium alloys, Biomaterials 30 (2009) 484-498.

[13] X.N. Gu, X.H. Xie, N. Li, Y.F. Zheng, L. Qin, In vitro and in vivo studies on a Mg–Sr
binary alloy system developed as a new kind of biodegradable metal, Acta Biomaterialia 8
(2012) 2360-2374.

[14] L. Hou, Z. Li , Y. Pan, L. Du , X. Li, Y. Zhenga, L. Li, In vitro and in vivo studies on
biodegradable magnesium alloy, Progress in Natural Science: Materials International 24
(2014) 466–471.

29
 ACCEPTED MANUSCRIPT

[15] L. Yang, Y. Huang, F. Feyerabend, R. Willumeit, C. Mendis, K.U. Kainer, N. Hort,

Microstructure, mechanical and corrosion properties of Mg–Dy–Gd–Zr alloys for medical
applications, Acta Biomaterialia 9 (2013) 8499-8508.

[16] J. Kubasek, D. Vojtech, Structural and corrosion characterization of biodegradable Mg–

RE (RE=Gd, Y, Nd) alloys, Transactions of Nonferrous Metals Society of China 23 (2013)
1215-1225.

T
IP
[17] N. Hort, Y. Huang, D. Fechner, M. Stormer, C. Blawert, F. Witte, C. Vogt, H. Drucker, R.
Willumeit, K.U. Kainer, F. Feyerabend, Magnesium alloys as implant materials – Principles of
property design for Mg–RE alloys, Acta Biomaterialia 6 (2010) 1714-1725.

R
[18] N. Angrisani, J. Reifenrath, J.-M. Seitz, A. Meyer-Lindenberg, Rare Earth Metals as

SC
Alloying Components in Magnesium Implants for Orthopaedic Applications, Materials
Science, Metals and Nonmetals, "New Features on Magnesium Alloys", Chapter 4, 2012, 1-
22.

NU
[19] G.E. Jai Poinern, S. Brundavanam, D. Fawcett, Biomedical Magnesium Alloys: A Review
of Material Properties, Surface Modifications and Potential as a Biodegradable Orthopaedic
Implant, American Journal of Biomedical Engineering 6 (2012) 218-240.
MA
[20] X.-N. Gu, Y.-F. Zheng, A review on magnesium alloys as biodegradable materials,
Frontiers of Materials Science in China 4 (2010) 111-115.

[21] P. Vostrý, B. Smola, I. Stulíková, F. von Buch, B. L. Mordike, Microstructure Evolution in

D

Isochronally Heat Treated Mg–Gd Alloys, PHYSICA STATUS SOLIDI A-APPLIED

TE

RESEARCH 175 (1999) 491 - 500.

[22] A. Burmester, B. Luthringer, R. Willumeit, F. Feyerabend, Comparison of the reaction of

bone-derived cells to enhanced MgCl2-salt concentrations, Biomatter 4 (2014) 1-11.
P

[23] J. Fischer, D. Pröfrock, N. Hort, R. Willumeit, F. Feyerabend, Improved cytotoxicity

CE

testing of magnesium materials, Materials Science and Engineering B 176 (2011) 830–834.

[24] M. Hampl, C. Blawert, M.R. Silva Campos, N. Hort, Q. Peng, K.U. Kainer, R. Schmid-
AC

Fetzer, Thermodynamic assessment and experimental study of Mg–Gd alloys, Journal of

Alloys and Compounds 581 (2013) 166–177.

[25] Y.H. Qiuming Peng, J. Meng, Y. Li, K.U. Kainer, Strain induced GdH2 precipitate in Mg–
Gd based alloys, Intermetallics 19 (2011) 382–389.

[26] Z. Zhen, T.-f. Xi, Y.-f. Zheng, A review on in vitro corrosion performance test of
biodegradable metallic materials, Transactions of Nonferrous Metals Society of China 23
(2013) 2283-2293.

[27] N.I. Zainal Abidin, B. Rolfe, H. Owen, J. Malisano, D. Martin, J. Hofstetter, P.J.
Uggowitzer, A. Atrens, The in vivo and in vitro corrosion of high-purity magnesium and
magnesium alloys WZ21 and AZ91, Corrosion Science 75 (2013) 354–366.

[28] J.-C. Gao, S. Wu, L.-Y. Qiao, Y. Wang, Corrosion behavior of Mg and Mg-Zn alloys in
simulated body fluid, Transactions of Nonferrous Metals Society of China 18 (2008) 588-592.

[29] J. Hofstetter, M. Becker, E. Martinelli, A.M. Weinberg, B. Mingler, H. Kilian, S.

Pogatscher, P.J. Uggowitzer, J.F. Löffler, High-Strength Low-Alloy (HSLA) Mg–Zn–Ca Alloys
with Excellent Biodegradation Performance, Jom 66 (2014) 566-572.

30
 ACCEPTED MANUSCRIPT

[30] Z.G. Huan, M.A. Leeflang, J. Zhou, L.E. Fratila-Apachitei, J. Duszczyk, In vitro
degradation behavior and cytocompatibility of Mg–Zn–Zr alloys, Journal of materials science.
Materials in medicine 21 (2010) 2623-2635.

[31] D. Hong, P. Saha, D.-T. Chou, B. Lee, B.E. Collins, Z. Tan, Z. Dong, P.N. Kumta, In vitro
degradation and cytotoxicity response of Mg–4% Zn–0.5% Zr (ZK40) alloy as a potential
biodegradable material, Acta Biomaterialia 9 (2013) 8534–8547.

T
IP
[32] W.H. Ma, Y.J. Liu, W. Wang, Y.Z. Zhang, Improved biological performance of
magnesium by micro-arc oxidation, Brazilian journal of medical and biological research 48
(2015) 214-225.

R
[33] A.E. Coy, F. Viejo, P. Skeldon, G.E. Thompson, Susceptibility of rare-earth-magnesium

SC
alloys to micro-galvanic corrosion, Corrosion Science 52 (2010) 3896-3906

[34] B. Ullmann, J. Reifenrath, J.M. Seitz, D. Bormann, A. Meyer-Lindenberg, Influence of the

NU
grain size on the in vivo degradation behaviour of the magnesium alloy LAE442, Proceedings
of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine 227
(2013) 317-326.
MA
[35] O. Charyeva, F. Feyerabend, R. Willumeit, D. Zukowski, C. Gasqueres, G. Szakacs,
N.A. Agha, N. Hort, F. Gensch, F. Cecchinato, R. Jimbo, A. Wennerberg, K.S. Lips, In Vitro
Resorption of Magnesium Materials and its Effect on Surface and Surrounding Environment,
MOJ Toxicology 1 (2015).
D

[36] F. Witte, J. Fischer, J. Nellesen, H.A. Crostack, V. Kaese, A. Pisch, F. Beckmann, H.

TE

Windhagen, In vitro and in vivo corrosion measurements of magnesium alloys, Biomaterials

27 (2006) 1013-1018.

[37] D. Xue, Y. Yun, Z. Tan, Z. Dong, M.J. Schulz, In Vivo and In Vitro Degradation Behavior
P

of Magnesium Alloys as Biomaterials, J. Mater. Sci. Technol. 28 (2012) 261–267.

CE

[38] J. Walker, S. Shadanbaz, N.T. Kirkland, E. Stace, T. Woodfield, M.P. Staiger, G.J. Dias,
Magnesium alloys: Predicting in vivo corrosion with in vitro immersion testing, Journal of
biomedical materials research Part B: Applied biomaterials 100B (2012) 1134-1141.
AC

[39] S S. Shadanbaz, J. Walker, T.B. Woodfield, M.P. Staiger, G.J. Dias, Monetite and
brushite coated magnesium: in vivo and in vitro models for degradation analysis, Journal of
materials science. Materials in medicine 25 (2013) 173-183.

[40] W.D. Müller, M.L. Nascimento, M. Zeddies, M. Córsico, L.M. Gassa, M.A. Fernández
Lorenzo de Mele, Magnesium and its Alloys as Degradable Biomaterials. Corrosion Studies
Using Potentiodynamic and EIS Electrochemical Techniques, Materials Research 10 (2007)
5-10.

[41] X.N. Gu, N. Li, Y.F. Zheng, L. Ruan, In vitro degradation performance and biological
response of a Mg–Zn–Zr alloy, Materials Science and Engineering: B 176 (2011) 1778-1784.

[42] R. Willumeit, J. Fischer, F. Feyerabend, N. Hort, U. Bismayer, S. Heidrich,B. Mihailova,

Chemical surface alteration of biodegradable magnesium exposed to corrosion media, Acta
biomaterialia 7 (2011) 2704–2715.

[43] N.A. Agha, F Feyerabend, B Mihailova, S Heidrich, R Willumeit, Magnesium corrosion

under physiological conditions, European Cells and Materials 28 (2014) 22.

31
 ACCEPTED MANUSCRIPT

[44] L. Yang, N. Hort, R. Willumeit, F. Feyerabend, Effects of corrosion environment and

proteins on magnesium corrosion, Corrosion Engineering, Science and Technology 47
(2012) 335-339.

[45] N. Eliaz, Degradation of Implant Materials, Springer Science and Business Media, New
York, 2012.

T
[46] R. Willumeit-Römer, H.-P. Wendel, B. Mihailova, N.A. Agha, F. Feyerabend, How does

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blood contact change Magnesium degradation?, European Cells and Materials 28 (2014) 29.

R
SC
NU
MA
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P TE
CE
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Highlights

 Viable pre-osteoblast cells verified for 13 days on the Mg alloys in in vitro conditions.
 Degradation performance of pure Mg, Mg2Ag and Mg10Gd compared in in vitro and

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in vivo conditions.
 Mg2Ag reveals a significant higher in vitro degradation rate after one week in

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comparison to the in vivo situation, whereas after 4 weeks degradation rates of the
materials are comparable between in vitro and in vivo conditions.

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