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In vivo study of a biodegradable orthopedic screw (MgYREZr-alloy) in a rabbit

model for up to 12 months

Article in Journal of Biomaterials Applications · January 2013

DOI: 10.1177/0885328212472215 · Source: PubMed

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Article
Journal of Biomaterials Applications
2014, Vol 28(5) 667–675
! The Author(s) 2012
In vivo study of a biodegradable Reprints and permissions:
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orthopedic screw (MgYREZr-alloy) in DOI: 10.1177/0885328212472215
jba.sagepub.com
a rabbit model for up to 12 months

Hazibullah Waizy1, Julia Diekmann1, Andreas Weizbauer1, Janin Reifenrath2,

Ivonne Bartsch1, Volkmar Neubert3, Robert Schavan4 and Henning Windhagen1

Abstract
Biodegradable magnesium-based implants are currently being developed for use in orthopedic applications. The aim of
this study was to investigate the acute, subacute, and chronic local effects on bone tissue as well as the systemic reactions
to a magnesium-based (MgYREZr-alloy) screw containing rare earth elements. The upper part of the screw was
implanted into the marrow cavity of the left femora of 15 adult rabbits (New Zealand White), and animals were
euthanized 1 week, 12 weeks, and 52 weeks postoperatively. Blood samples were analyzed at set times, and radiographic
examinations were performed to evaluate gas formation. There were no significant increased changes in blood
values compared to normal levels. Histological examination revealed moderate bone formation with direct
implant contact without a fibrous capsule. Histopathological evaluation of lung, liver, intestine, kidneys, pancreas, and
spleen tissue samples showed no abnormalities. In summary, our data indicate that these magnesium-based screws
containing rare earth elements have good biocompatibility and osteoconductivity without acute, subacute, or chronic
toxicity.

Keywords
In vivo, biodegradation, magnesium alloy, orthopedic screw

The present study investigated the local and systemic

Introduction
The ideal biodegradable implant should have good bio-
compatibility, acceptable initial stability, and should
degrade progressively as new bone tissue regenerates.
Biodegradable polymers such as PLLA, PLG, and
PLGA are currently in use clinically. However, the
mechanical properties of the polymers, as well as local
inflammatory effects, limit their use.1 In recent years
research become more focused on magnesium alloys as
degradable implants for orthopedic applications.2,3
Magnesium and magnesium alloys are biodegradable,
have positive effects on bone remodeling, and have desir-
able mechanical properties, such as high tensile strength
and a favorable Young’s modulus (E ¼ 41–45 GPa).4
Both of the latter values are closer to those of natural
bone compared to metallic implants currently in use and
may therefore minimize stress-shielding.3,4 The osteo-
conductive effect of magnesium alloys is considered
beneficial for orthopedic applications.5,6 But magne-
sium-based implants used to have high corrosion rates
accompanied with hydrogen gas production.2
effects of a magnesium-based screw, which is made of
an MgYREZr magnesium alloy that contains rare earth
elements (REE) and is similar to the WE43 alloy.
Several studies have already investigated the orthopedic
use of rare earth-containing magnesium alloys such as
LAE442 or WE43, and good biocompatibility has been
reported in vivo.5,7–9 However, the biocompatibility of
REE is still controversial, on the one hand these elem-
ents are suspected to have some adverse effects,10–12
on the other hand other authors reported good
1
Department of Orthopaedic Surgery, Hannover Medical School,
Hannover, Germany
2
Small Animal Clinic, University of Veterinary Medicine Hannover,
Hannover, Germany
3
Institute for materials testing and materials engineering Dr. Neubert
GmbH, Clausthal-Zellerfeld, Germany
4
Syntellix AG, Schiffgraben 11, Hannover, Germany
Corresponding author:
Hazibullah Waizy, Department of Orthopaedic Surgery, Hannover
Medical School, Anna-von-Borries-Str. 1-7, 30625 Hannover, Germany.
Email: hazibullah.waizy@ddh-gruppe.de

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668 Journal of Biomaterials Applications 28(5)

biocompatibility of rare earth metals.13,14 The aim of

this study was to investigate the acute, subacute, and
chronic local effects of the MgYREZr screw on bone
tissue as well as the systemic effects using an in vivo
animal model.

Materials and methods

Implants
The cannulated screws had a total length of 20 mm, a
shaft diameter of 2.0 mm and a bore diameter of
1.3 mm. The design of the screw includes two threads
with different pitches (3.0 and 4.0 mm) to gain inter-
fragment compression. The implants are made of a
powder metallurgically processed magnesium alloy.
This aluminum-free material consisted of MgYREZr,
which is similar to WE43, and contains more than
90 wt% magnesium. With an average grain size less
than 5 mm, this high performance alloy exhibits an
offset elastic limit of Rp0.2>250 MPa, a tensile strength
>275 MPa, and elongation at break of more than 10%.
Animal experiments and surgery
The animal experiments were conducted using a
protocol that was approved by the ethics committee
and that complied with German animal welfare legis-
lation (Approval No. 33.12-42502-04-10/0187). Fifteen
adult female New Zealand White Rabbits (mean body
weight of 3.9  0.3 kg) were randomly assigned into
three experimental groups of 5 animals each: 1 week
(group 1), 12 weeks (group 2) and 52 weeks (group 3).
Surgery was performed under general anesthesia that
was induced by intramuscular injection with 25 mg/kg
ketamine, 2 mg/kg midazolam and 0.1 mg/animal gly-
copyrroniumbromide. For analgesia, the rabbits were
given preoperative 0.15 mg/animal buprenorphin and
4 mg/kg carprofen. To perform the endotracheal
intubation, each animal was injected intravenously
with 1 mg/kg propofol. The anesthesia was maintained
by isoflurane delivered in oxygen (1.5–3.5% oxygen
mixture; 1.5 L/min). Infusion of Ringer’s lactate solu-
tion (10 mL/kg/h) during the entire surgical procedure
ensured cardiovascular function. In each animal the
upper part of 1 screw (diameter: 3 mm; length:
6 mm) (Figure 1) was inserted into the supracondylar
region of the left femur. At the end of each investiga-
tion period, all animals were euthanized by intraven-
ous overdose of pentobarbital and the femur was
harvested directly after sacrifice and fixated in 4%
formaldehyde for postmortem study by micro-com-
puted tomography (micro-CT) and histological ana-
lysis. One rabbit in group 2 died while sedated for
postoperative radiographs, and one rabbit in group 1
Figure 1. An example of the magnesium-based screw used in
this study. The tip of the screw (diameter: 3 mm; length: 6 mm)
was inserted into the left femur of each rabbit.
was sacrificed the day after surgery because of a frac-
ture of the operated hind limb.
Postoperative observation and radiographic
evaluation
Animals were observed daily during the first three
weeks and afterwards every third day to investigate
the general condition, possible gas cavities or inflam-
mations. Radiographs were performed (anterior-pos-
terior view and lateral view) in all animals
immediately after surgery to verify implant position.
Additional radiographs were performed after surgery
(1 week, 3 weeks, 6 weeks, 12 weeks, 26 weeks, 39
weeks, and 52 weeks) to observe the degradation pro-
cess and to detect feasible formation of gas cavities.
Blood analysis and histopathology
Blood samples were collected and analyzed at set time
points according to ISO10993-11 to determine the
serum magnesium ion concentrations and to observe
whether there was a systemic inflammatory reaction.
The blood samples were analyzed by a certified veter-
inary diagnostic laboratory (Laboklin GmbH & Co.
KG, Bad Kissingen, Germany). Histopathological ana-
lysis of samples of lung, liver, intestine, kidneys, pan-
creas, and spleen tissues was performed to monitor
systemic toxicology at different times after implantation
by a certified veterinary diagnostic laboratory
(Laboklin GmbH & Co. KG, Bad Kissingen,
Germany). The tissues were embedded in paraffin wax
and 3 mm thick sections were cut and stained with

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Waizy et al.
hematoxylin-eosin. Examination was ensued
microscopy.
mCT
The in vivo biodegradation of the implants in the femur
was studied using mCT (Micro-CT 80 system,
SCANCO MEDICAL, Brüttisellen, Switzerland) with
a scan resolution of 36 mm. The system was operated at
55 kV, with a current of 145 mA and an integration time
of 600 ms. Per sample 299-355 slices were taken. The
evaluation was performed using the MICRO-CT
Evaluation Program V6.0 (SCANCO MEDICAL,
Brüttisellen, Switzerland) by manually contouring the
surface of the screw. The threshold for all samples was
defined using a value of 285. For the analysis of the
density, the results were compared to a nondegraded
screw.
Histology
Histological analysis of bone samples from each animal
was performed at a certified laboratory that is specia-
lized in bone histology (HIK-laboratory, Hannover,
Germany). The samples were embedded in methyl-
methacrylate (Technovit 9100 NewÕ , Heraeus Kulzer,
Hanau, Germany) according to established protocols.
Three specimens per animal were chosen for further
analysis by toluidine blue staining, 40 mm sections of
bone that were cut perpendicular to the implants were
prepared using the cutting-grinding technique and then
polished.15
Toluidine blue staining. Toluidine blue staining was per-
formed using two solutions. Solution A contained
sodium tetraborate (Merck, Darmstadt, Germany)
and toluidine blue (WALDECK GmbH & Co
Division Chroma, Münster, Germany), and solution
B contained Pyronin G (Merck, Darmstadt,
Germany). Solutions A and B were mixed and then
filtered. The sections were incubated in the freshly
mixed solution for 15 min; after drying, sections were
mounted on coverslips (Heraeus Kulzer, Hanau,
Germany).
Microscopy
Sections were examined by light microscopy to obtain a
qualitative impression of any major changes, with a
focus on the number of inflammatory cells and on the
area where the bone contacted the implant. Images
were taken at 10  magnification. A circle with a
radius of 2 mm around the center of the implant was
chosen as the region of interest (ROI). Within each
ROI, two sub-regions were examined further. One of
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669
by the regions was the direct interface between the implant
and the bone tissue, and the other region was the adja-
cent tissue, which we called the ‘surrounding tissue’.
Each qualitative evaluation resulted in assigning each
sample a score, using a modified version of the scoring
system proposed16 (Table 1).
Energy-dispersive X-ray analysis
A sample after 52 weeks implantation was cut as for
histological analysis, and the surface was coated with
gold before energy-dispersive X-ray analysis (EDX)
was performed to detect the elements on the surface.
An area of the screw was analyzed as well as the embed-
ding medium.
Statistical analysis
All statistical evaluations were performed with
ANOVA. Statistically significant differences were
defined as p < 0.05.
Results
Clinical and radiographic results
In the first few days after surgery, the rabbits showed
normal postoperative appearance of a relatively slight
wound swelling and reddening. All clinical signs
resolved within 1 week after surgery. The animals
regained full weight-bearing use of the implanted leg.
Further clinical signs for local inflammatory reactions
or suture intolerance were not present during the
remaining implantation period. In addition, there was
no formation of gas cavities that could be detected
through observation and palpation. The native radio-
graphic examination showed no obvious signs of gas
cavity formation or bone destruction.
Blood analysis and histopathology
Blood analyses were performed before implantation of
the screws and at defined times after implantation
(Figure 2). The blood sample analyses showed no
values that were significantly increased in comparison
to normal values. Histopathological evaluation of
tissue samples displayed minor findings of degeneration
and hyperplasia in the liver and spleen that were most
likely due to anesthesia and surgery. Kidney tissue sam-
ples were normal.
mCT
Figure 3(a) to (c) shows the micro-CT pictures. The
analysis revealed a slight decrease in the average
670 Journal of Biomaterials Applications 28(5)

Inflammation
screw density compared to a nondegraded screw after
12 weeks implantation (95.5%) to 52 weeks after
implantation (group 3) (95.3%). There were no signifi-

0
cant differences between the groups. The drill hole in
the 7-week group is clearly visible and after 52 weeks of

Tissue other than bone (e.g., fibrous

implantation integration of the tip of the remaining
implant into the newly formed bone could be observed.
In the scans, the shape of the screw and its threads was
still visible 52 weeks after implantation (Figure 3(c)).
Fibrous tissue capsule

However, white areas on the mCT-scan of the screw

tissue) indicated material change.

Histological evaluation
The histological analysis of the bone samples are
1

shown in Figures 3(d) to (f) and 4. Initial degradation

Localized fibrous tissue not arranged

Lamellar or woven bone with bone-

of the thread was observed 1 week after implantation

forming activity and osteoclastic

(Figure 3(d)). After 12 weeks, the degradation process

Table 1. Histological evaluation of bone samples using a scoring system modified from the methods of An and Martin.16

was more advanced, with about 50% of the metallic

part of the implant degraded. Moderate apposition of
the bone was observed in this group in direct contact
with the implant (Figure 3(e), Table 2). The metallic part
as a capsule

of the implant appeared to be fully degraded 52 weeks

activity

after implantation (Figure 3(f)). The formation of bone

trabeculae from the corticalis to the implant was observed
after 52 weeks of implantation (Figure 4(a)). Moderate
2

bone formation around the implant remaining could be

seen (Figure 4(a) and (d)). Newly formed bone appear
Lamellar or woven bone with bone-
Remodeling lacuna with osteoblasts

to be grown into parts of the degraded material

and/or osteoclasts at surface

(Figure 4(e)). It was apparent that most of the specimens

in the 52 weeks group showed direct contact of newly
forming activity

Table 2. Histological analysis of bone samples at the indicated

time points after screw implantation.
Percentage of the score value
concerning
3

Score Surrounding
Score value and evaluation parameter

Group value Interface tissue

without soft-tissue interlayer

Group 1 (n ¼ 4) 0 50 0
Similar to original cortical bone
Direct bone to implant contact

1 week 1 17 100
2 33 0
3 0 0
4 0 0
Group 2 (n ¼ 4) 0 0 0
12 weeks 1 0 42
2 42 8
3 0 33
4

4 58 17
Surrounding tissue

Group 3 (n ¼ 5) 0 0 0
52 weeks 1 7 47
2 13 7
Interface

3 33 27
4 47 20

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Waizy et al. 671

formed bone with the implant (Figure 4(f)). A few of the In vivo studies are needed to determine the inter-
specimens showed fibrous tissue in the region surround- action of implants with the surrounding blood and
ing the implant, but only one of the specimens in the body fluids. Blood composition was analyzed to
chronic group (group 3) showed slight formation of a assess the level of released Mg2þ ions. We found no
defined fibrous structure (Figure 4(c)). significant elevation of the serum magnesium level com-
pared to the normal values. The regulation of magne-
sium takes place in the kidneys, which reabsorb Mg2þ
EDX analysis and excrete the excess via urine.17,18 Biochemical exam-
The results of the EDX analysis are shown in Figure 5. ination showed that the tested levels of serum magne-
The analyzed surface of the remaining implant consisted sium and creatinine were within the reference ranges in
mainly of C, Ca, P, and O, with a minor presence of all animals. Furthermore, histopathological examin-
Yttrium and REE. Notably, the analysis revealed ation of the kidneys showed no pathologies. These
barely any Mg. In comparison the EDX analysis of the observations are in accordance with other studies
cannulated part of the screw showed high content of where no increase in serum magnesium or result in
carbon and oxygen. This area should mainly represent kidney disorders were found.7,18,19
the embedding medium polymethylmethacrylate The MgYREZr-based screw used in this in vivo
(Technovit 9100). study contains small quantities of REE. These alloying
elements help to improve the mechanical properties and
corrosion resistance of the implants.3 In addition to
Discussion
their positive properties, REE also have some known
The aim of the present study was to investigate the local negative effects. Chelated REE are excreted mainly via
and systemic effects of a biodegradable magnesium- the urine after transient accumulation in the kidneys
based implant containing REE after three defined with a half time of several hours. Rare earth chlorides,
implantation periods. which are a product of corrosion, are taken up by the

(a) 1.8 (b) 170

160
Serum magnesium (mmol/L)

1.6
Serum Creatinine (mmol/L)

150
140
1.4 130
120
1.2 110
1 100
90
0.8 80
70
0.6 60
50
0.4 40
30
0.2 20
10
0 0
0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50
Time (weeks) Time (weeks)

(c) 12 (d) 60
GPT
10 50 GOT
Serum UREA (mmol/L)

GPT and GOT (Ul/L)

8 40

6 30

4 20

2 10

0 0
0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50
Time (weeks) Time (weeks)

Figure 2. Results of the blood biochemical analysis at the indicated time points after screw implantation: (a) serum magnesium (b)
serum creatinine (c) serum UREA (d) GPT (glutamic pyruvic transaminase) and GOT (glutamic oxaloacetic transaminase). The dashed
line marks the recommended level. The data are expressed as mean  standard deviation.

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672 Journal of Biomaterials Applications 28(5)

Figure 3. a)–(c): Micro-CT of implants in the marrow cavity of the rabbit femora. (a) 1 week, (b) 12 weeks, and (c) 52 weeks after
implantation. The contours of the screw were visible in all scans. (d)–(f): Histological analysis of toluidine blue stained bone samples at
the indicated times after screw implantation. (d) 1 week after implantation, the metallic part of the magnesium screw is clearly visible.
Initial degradation of the thread can be seen (black arrow). (e) 12 weeks after implantation, the screws show more advanced
degradation. Sparse bone apposition was observed. (f) The metallic part of the implant appears to be fully degraded 52 weeks after
implantation. We assume that there is an apatite construct remaining, with apposition of bone around the implant.

Figure 4. Histological analysis of the samples with toluidine blue staining after 52 weeks of implantation. Moderate bone formation
around the implant remaining could be observed with the formation of bone trabeculae from the corticalis to the implant. Direct
bone/implant contact was found (f). The presence of small parts of fibrous tissue was observed at higher magnification in some samples
(c).

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Waizy et al. 673

liver and spleen and are not easily excreted.11 Thus,

Figure 5. EDX analysis of the sample 52 weeks after implantation. The black box on the SEM image indicates the area that was analyzed: (a) area of the embedding medium
biodegradation of implants containing REE might
have adverse effects in these organs Haley investigated
the toxicology and pharmacology of several REE by
intravenous injection of the salts of REE.10 Haley cre-
ated a worst-case scenario, with high serum levels of
REE that led to an increased concentration in the
organs and resulted in organ degeneration and fatty
liver. In contrast to Haley’s findings, our histopatho-
logical findings were nonspecific. We assume that the
underlying cause of the liver degeneration we observed
was hypoxia and the effects of the anesthetic agents. In
summary, the results of blood analysis and histopatho-
logical examination lead us to conclude that the deg-
radation of this magnesium alloy caused no acute,
subacute, or chronic systemic inflammation reactions
or pathological changes in the visceral organs. This
indicates good in vivo systemic biocompatibility.
The first magnesium-based implants were limited by
high corrosion rates and by generation of subcutaneous
gas cavities by hydrogen gas development.20 The cor-
rosion of 1 g of pure magnesium produces 1 l of hydro-
gen gas. Several studies describe the occurrence of gas
formation by implant degradation.5,21 In an in vivo
study, Li et al. observed gas shadows in soft tissue
and bone marrow cavity around the MgCa0.8 implant
during the early implantation period. The gas dis-
appeared two months after implantation, and no
adverse effects were detected.21 This observation is con-
sistent with other studies.22,23 Zhang et al.23 showed
EDX: energy-dispersive X-ray spectroscopy; SEM: scanning electron microscopy.

that the subcutaneous gas bubbles generated by a

Mg-6Zn alloy disappeared 6 weeks after implantation,
while Hänzi et al.22 reported limited gas formation by a
WE43 implant after 91 days. In the current study, we
examined possible gas cavities by clinical observation
and by performing x-rays at defined time points and
observed no gas formation within the soft tissue. Our
(cannulated part of the screw) and (b) area of the screw.

observations are consistent with earlier studies of AX30

and LANd442.24,25
The mCT findings in this study showed a slight
decrease of the screw density compared to the noncor-
roded screw. The mCT-scans revealed the shape of the
screw and its thread which are visible even after 52
weeks implantation time (Figure 3). These observations
are in contrast with those in the histological evaluation.
Specifically, this examination indicated that nearly 50%
of the metallic part of the implant was degraded after 3
months, and the metallic part of the screws appeared to
be fully degraded after 52 weeks (Figure 3). The EDX
analysis of the sample supported these observations, as
nearly no Mg was detected 52 weeks after implantation
(Figure 5). Rather, EDX analysis showed mainly C, Ca,
P, and O, with minor amounts of REE, at the 52 weeks
point in time. We suppose that 52 weeks after implant-
ation, the remaining implant consists mainly of

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674
substances similar to an apatit formation. We assume
that the carbon content is attributed to the embedding
medium, which consisted of polymethylmethacrylate.
The EDX analysis of the cannulated part of the
screw, which represented mainly the embedding
medium, supported this assumption. In this analysis
high content of carbon and oxygen were found. The
gold peaks were due to the sample preparation process
for EDX analysis.
Most studies suggest that magnesium alloys confer
enhanced bone bonding to implant surfaces compared
to conventional materials.6,7,26 New bone formation
has been described for MgCa0.8, AX30, LANd 442,
ZEK100, WE43, and LAE442.2 Reifenrath et al.27
also showed a periosteal increase in the mineral appos-
ition rate, which was calculated after intravital fluores-
cent labeling. Castellani et al.7 observed greater bone/
implantcontact with a magnesium alloy based on WE43
compared to a titanium implant. Witte et al.5 examined
four different magnesium alloys and reported a greater
mineral apposition rate compared to a degradable poly-
mer. These results are in agreement with our findings.
In some specimens, there is a visible appearance of
fibrous tissue, but only in one case was there any evi-
dence of the formation of a defined fibrous structure.
We observed moderate bone apposition to the implant,
with direct contact to the screw (Figures 3 and 4). The
cracks of the remaining implant material might be
caused by cutting processes due to histological sample
preparation (Figure 4). The presence of osteoclasts and
osteoblasts also indicated an ongoing bone remodeling
process. These observations support the idea that the
MgYREZr screw has an osteoconductive capacity and
suggest good biocompatibility.
Conclusions
This study showed that implantation of the magne-
sium-based screw, which is composed of a magnesium
alloy (MgYREZr) and contains rare earth elements,
resulted in no significant changes in blood values and
in moderate bone formation with direct bone/implant
contact. There was no development of a fibrous cap-
sule. All organs appeared normal. In summary, these
results indicate good biocompatibility and osteocon-
ductivity of the MgYREZr screw without acute, sub-
acute, or chronic toxic effects.
Acknowledgments
We thank Sylvie Titze, Ulrike Kreimeyer, Patrick Helmecke,
and Maren Cassens for excellent technical support. We thank
Christopher Müller for creating Figure 1.
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Journal of Biomaterials Applications 28(5)
Funding
This study was partially financed by Syntellix AG, Hannover,
Germany.
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