in vivo evaluation of degradable magnesium alloys as ... · protection by the use of high purity...
Post on 26-May-2020
4 Views
Preview:
TRANSCRIPT
Klinik für Kleintiere
Stiftung Tierärztliche Hochschule Hannover
In vivo evaluation of degradable magnesium alloys
as orthopedic implant material in suitable animal
models
Habilitationsschrift
Zur Erlangung der Venia Legendi
An der Tierärztlichen Hochschule Hannover
Janin Reifenrath
Hannover, 2015
Tag der „nichtöffentlichen wissenschaftlichen Aussprache“: 11.11.2015
ZITAT
„Wissenschaft ist nur der Austausch unserer Unwissenheit gegen
Unwissenheit neuer Art.“
Lord George Gordon Noel Byron (1788 – 1824)
Contents
1 Preface 5
2 Abbreviations 6
3 Introduction 7
3.1 Implant materials for orthopedic applications 7
3.1.1 Common materials 7
3.1.2 Magnesium alloys 8
3.2 Evaluation of degradation and biocompatibility of magnesium based
implants 13
3.2.1 In vitro methods for degradation and toxicology evaluation 13
3.2.2 In vivo examination of magnesium based implant materials for
orthopedic use 14
3.2.2.1 Suitable animal models for the examination of new orthopedic
implant materials 15
3.2.2.2 Evaluation methods for degradation analysis and biocompatibility
in vivo 16
3.3 Simulation models as an option for reducing animal experiments 19
4 List of Publications (contributing to the current work) 21
5 Results and Discussion 25
5.1 In vivo degradation characteristics and biocompatibility of different
magnesium based alloys in contrast to conventional implant
materials 25
5.2 Influence of handling and storage on magnesium based implants 40
5.3 Application-oriented complex mg-based implant materials
(plate-screw-systems and intramedullary nailing systems) for
fracture fixation in weight bearing bones 42
5.4 Biomechanical implant requirements for fracture fixation in weight
bearing bones 51
5.5 Comprehensive discussion of used animal models for the
investigation of biomaterials for orthopedic applications 54
6 Summary 59
7 References 61
8 Presentation of the own work 77
9 Acknowledgement 87
1. Preface
Implant materials are commonly used in orthopedic surgery. While permanent implant
materials e.g. for total joint replacement are designed to remain in the body as long as
possible, temporary implants e.g. for fracture fixation are removed after healing. Until
today, predominantly surgical steel, titanium and cobalt chromium alloys are used. To
avoid a second surgery for implant removal, degradable implant materials are an
alternative approach for temporary implants. However, available degradable materials
like polymers are not stable enough for fracture fixation in weight bearing bones.
Therefore, biodegradable magnesium alloys are promising materials.
In general, the development of new materials for clinical applications contains previous
in vitro and in vivo studies. For this purpose, animal models are irreplaceable to
evaluate implant degradation and biocompatibility prior to clinical studies. The
presented work in this manuscript presents a summary of an interdisciplinary
collaborative research on the development of magnesium-based implant materials for
loaded applications in different animal models and is performed in cooperation with
material and engineering scientists, bio-mechanists, and orthopedic surgeons.
2. Abbreviations
PLLA poly-L-lactide-acid
PGA polyglycolid-acid
bw body weight
µ-CT micro-computer tomography
wt% weight percent
LAE442 magnesium alloy with 4wt% lithium, 4wt% aluminum
and 2wt% rare earth metals
Y yttrium
Z zinc
NOAEL no-observable-adverse-effect-level
ZEK100 magnesium alloy containing 1 wt% zinc and less
than 1 wt% rare earths and zirconium
UN/WHO United Nations/ World Health Organisation
AX30 magnesium alloy containing 3 wt% aluminum and
less than 1 wt% calcium
MgCa0.8 / MgCa1.0 magnesium alloys containing 0.8 wt%/ 1.0 wt%
calcium
WE43 magnesium alloy containing 4 wt% yttrium and 3 wt%
rare earths
MgGd magnesium alloy containing gadolinium
LANd442 magnesium alloy containing 4wt% lithium, 4wt%
aluminum and 2wt% neodymium
AZ91/ AZ31/ AZ63 magnesium alloy containing 9wt% / 3 wt% / 6 wt%
aluminum and 1wt% / 3 wt% zinc
LACer442 magnesium alloy containing 4wt% lithium, 4wt%
aluminum and 2wt% cerium
MgZK60 magnesium alloy containing 6 wt% zinc and less
than 1 wt% zirconium
MgZn magnesium zinc alloy
MgYZ magnesium, yttrium, zinc alloy
7
3. Introduction
3.1. Implant materials for orthopedic applications
3.1.1. Common materials
The most used implant materials are titanium and surgical steel and correspondent
alloys. In fracture fixation they are used as screws, osteosynthesis plates or
intramedullary nails (STIFFLER 2004; MILLER a. GOSWAMI 2007; DÉJARDIN et al.
2012) and result in satisfactorily bone healing with well clinical acceptance in human
and veterinary medicine (STIFFLER 2004). Nevertheless several negative aspects,
such as stress shielding (UHTHOFF a. FINNEGAN 1983; CHAO et al. 1989;
LÅFTMAN et al. 1989; NAGELS et al. 2003, NAGELS et al. 2003), soft tissue irritation
and inflammatory osteolysis caused by released toxic alloying particles as well as
implant loosening, can occur (SALEH et al. 2004; STIFFLER 2004). Compared to
surgical steel, titanium has half the weight, a higher elasticity, improved corrosion
resistance, less known allergic potential (SCHATZKER a. HOULTON 2002) and
therewith the need of a second surgery for implant removal is reduced, but still present.
An alternative approach for temporary implants is the use of resorbable materials. The
proceeding degradation of the material avoids the need for implant removal and
reduces biomechanical stress-shielding in a later stage of bone healing. Different
bioresorbable materials, predominantly polymers like poly-L-lactide-acid (PLLA) and
polyglycolid-acid (PGA), are already developed for medical application (ENGELBERG
a. KOHN 1991; MAJOLA et al. 1992; ROKKANEN et al. 2000). However, the
mechanical strength had to be improved for the use in fracture fixation of
biomechanical high loaded bones (GOGOLEWSKI 2000; BERRY 2008). Whereas
polylactid implants possess elastic moduli (tension) between 5 and 14 GPa depending
on the production process, which is less than values for natural bone (7-40 GPa,
8
(GOGOLEWSKI 2000), conventionally used permanent implant materials like titanium
(110 GPa) and surgical steel 316L (193 GPa) are considerably stiffer (PIENKOWSKI
et al. 1998; TSCHEGG et al. 2011). Therefore research in the field of degradable
materials presently investigates in different concepts. One research approach focuses
on the development of more stable copolymers or fiber reinforced materials for
osteosynthesis (LANDES et al. 2013). The potential use of fiber reinforced copolymers
in craniofacial surgery has already been proved experimentally (CHEN et al. 2013).
The degradation process in polymers proceeds by hydrolysis and a local pH decrease
due to acidic breakdown products (IGNATIUS a. CLAES 1996). During this
degradation period, cellular reactions with involvement of macrophages and foreign
body giant cells are described, which are mostly without clinical problems and
disappear after complete removal of the degradation products (BÖSTMAN a.
PIHLAJAMÄKI 2000; GOGOLEWSKI 2000). Nevertheless, a local inflammation is not
desired and less reactive materials would be advantageous.
A second research approach is the development of degradable magnesium based
implant materials. Whereas pure magnesium possesses an elastic modulus of
approximately 45 GPa, alloying of other elements improves strengths (AVEDESIAN
1999). These materials are more stable than polymers (HOFMANN 1995; KAESE
2002; STAIGER et al. 2006) and showed best mechanical properties compared to self-
reinforced polymer and titanium in experimental push-out tests (TSCHEGG et al. 2011)
as well as promising results concerning the biocompatibility in in vivo studies (WITTE
et al. 2007c; WALKER et al. 2014a).
3.1.2. Magnesium alloys
Magnesium is a widely distributed element in the natural world (WOLF a. CITTADINI
2003) and is incorporated in many biological functions e.g. as part of enzymes or
9
coenzymes, or for the neuromuscular transmittance of stimuli (TOPF a. MURRAY
2003). However, only 1% of the human body-magnesium circulates in the blood
plasma. Most amounts are accumulated in the bone, the liver and muscles. The
regulation is controlled by the kidney as excessive magnesium is excreted with the
urine. In general, it is described as relatively safe and assessed as non-toxic
(STAIGER et al. 2006).
First orthopedic applications of magnesium based implants were performed in the early
20th century (LAMBOTTE 1932; VERBRUGGE 1934; MCBRIDE 1938). While
LAMBOTTE (1932) and VERBRUGGE (1934) published successful use of magnesium
implants for fracture fixation with complete resorption and the appearance of harmless
volumes of gas, MCBRIDE (1938) observed that pure magnesium was not stable
enough. Therefore, he preferred aluminum and manganese containing alloys and
recommended them as acceptable for the use as bone screws and pegs (MCBRIDE
1938). Despite successful applications, magnesium implants fell into oblivion for many
years, which might be due to an uncontrolled corrosion or to the emerging of other
materials like surgical steel or titanium (DISEGI a. ESCHBACH 2000; POHLER 2000).
During the corrosion process of magnesium, gas formation was an obvious
phenomenon (MCBRIDE 1938; WITTE 2010). Whereas the corrosion process of
magnesium and its alloys depends on the exposure to a corrosive medium, in general
the following corrosion reaction describes the corrosion mechanism in an aqueous
environment (SONG a. ATRENS 1999):
Mg → Mg2+ + 2e- (anodic reaction)
2H2O + 2e- → H2 + 2OH- (cathodic reaction)
Mg + 2H2O → Mg(OH)2 + H2 (overall reaction)
Due to the described reaction, hydrogen gas as well as magnesium hydroxide is
formed (SONG a. ATRENS 2003). While magnesium hydroxide can act as a protective
10
layer under standard environmental conditions, in solutions, which were adapted to the
in vivo situations or in vivo itself, containing chloride ions lead to formation of the highly
soluble MgCl2, which promotes the corrosion process. Knowledge of special corrosion
protection by the use of high purity magnesium, alloying elements or coatings was very
rare and might have been the reason for the replacement by other materials in clinical
use.
Magnesium as alternative implant material started to regain popularity in the late
1990s. Since then, a huge amount of in vitro and in vivo studies were performed and
in the year 2014 first materials were accredited for the use in clinical studies in Europe
(MAGNEZIX® Compression Screw 3.2, Syntellix AG, Hannover, Germany) and South
Korea (U&I Corporation, South Korea). Nevertheless, there is still considerable
potential in the development of new alloys and designs for the use in many different
applications.
Beside an influence on degradation behavior, alloying of elements influences
mechanical characteristics. Common elements for this purpose are aluminum, zinc,
calcium, manganese and rare earth metals. Aluminum highly improves strengths and
hardness as well as castability. Zinc is often used in combination with zirconium and
rare earths to produce precipitation-hardenable magnesium alloys, having a good
strength and is next to aluminum in effectiveness as an alloying ingredient in
magnesium (AVEDESIAN 1999). Besides to mechanical influences, zinc reduces the
effect of possible iron and nickel impurities on corrosion processes (POLMEAR 1999).
Whereas calcium increases the corrosion resistance already in low concentrations (LI
et al. 2008), rare earth metals have beneficial effects on the castability, improve the
tensile and creep properties as well as the corrosion resistance by forming solid
solutions or intermetallic compounds (NAKATSUGAVA et al. 1998). Other used
elements are lithium, zirconium, silver and manganese (AVEDESIAN 1999). Lithium is
11
known to stabilize the corrosion layer by alkalizing it (WANG 1997) and zirconium
improves the corrosion resistance in magnesium alloys by forming complexes with zinc
and certain elements which are impurities whereas silver can increase tensile
properties (AVEDESIAN 1999).
Beside the positive mechanical and corrosive resistant effects, potential toxicity always
has to be taken into account (YUEN a. IP 2010). These potential toxic effects of alloying
elements highly depend on the reached local or systemic concentrations during the
degradation process and have to be considered in the evaluation of biocompatibility
studies.
While calcium as alloying element is an essential element in the human body and
naturally belongs to the bone (KANNAN a. RAMAN 2008) other elements might be
more problematic, at least in critical local or systemic concentrations. Lithium and rare
earth elements are mostly evaluated with a low systemic toxic potential (HIRANO a.
SUZUKI 1996; GRANDJEAN a. AUBRY 2009b). Lithium-carbonate is even used
therapeutically in manic-depressive psychosis (GRANDJEAN a. AUBRY 2009a)
although negative effects like gastrointestinal pain or discomfort, polyuria, negative
effects on memory, vigilance and reaction time were observed in daily intake up to
1300mg (GRANDJEAN a. AUBRY 2009b). Another study with rats could even find a
potential of lithium to reduce aluminum–induced cytotoxic effects in the brain (BHALLA
et al. 2010). Rare earths in the chelated form are rapidly excreted via urine and
therewith systemically less effective, whereas unchelated ionic rare earths easily form
colloid in blood and are taken up by phagocytic cells of the liver and spleen. In addition,
the bone is another target organ whose clearance is known to be very slow (HIRANO
a. SUZUKI 1996). Local toxic effects are not described yet, but cannot be completely
excluded. Even aluminum is an element, which accumulates in the bone and can cause
12
local effects like decreased bone formation and mineralization (MARTÍNEZ et al.
2011). Furthermore it is regarded as critical concerning systemic effects. WILLHITE et
al. (2012) postulated a no-observable-adverse-effect-level (NOAEL) of 13 mg/kg-day
as total Al which could be identified in a 7-year follow up osteomalacia study based on
histologic data of adult hemodialysis patients. The UN/WHO expert committee on food
additives, however, reduced the provisional weekly tolerable level for aluminum in
2007 from 7 mg Al/kg bw/week to 1 mg Al/kg bw/week (WORLD HEALTH
ORGANISATION 2007) and WALTON (2014) described aluminum contributions to
Alzheimer Disease neuropathology. Although systemic concentrations during a slow
implant degradation process are presumably very low, the possibility of toxic effects
should be taken into account.
Zirconium is widely accepted as biocompatible and already used in dental alloys and
relatively inert orthopedic implants (SALDAÑA et al. 2007; LEE et al. 2010). The daily
human uptake has been known to be as high as 125 mg, and toxic effects induced by
very high concentrations were non-specific in nature (GHOSH et al. 1992). In
magnesium alloys, an exposure limit for zirconium is not known yet (YUEN a. IP 2010).
However, in magnesium based implants predominantly less than 1 weight percent are
used (e.g. ZEK100) and toxic concentrations are unlikely.
There is a similar situation for zinc. While zinc overdoses can reduce the erythrocyte
superoxide dismutase level in high concentrations, the tolerable daily exposure level
with 0.83 mg/ kg bw (YUEN a. IP 2010) will not be reached in the common used alloys
with less than 6 wt% (XU et al. 2009; ZHANG et al. 2010). Even implants with higher
concentrations of up to 35 wt% zinc (ZBERG et al. 2009) were evaluated as
biocompatible after subcutaneous implantation.
3.2. Evaluation of degradation and biocompatibility of magnesium based implants
3.2.1. In vitro methods for degradation and toxicology evaluation
13
In general, new implant materials are first tested in vitro to assess information of
possible toxic effects or degradation characteristics prior to in vivo studies. These
approaches are also used for the evaluation of magnesium based alloys. Different in
vitro examinations in various synthetic mediums (simulated body fluids, NaCl-solution,
Hank`s solution, etc.) are described to examine the corrosion characteristics
(MUELLER 2007; PARDO et al. 2008; XU et al. 2008; HÄNZI et al. 2009; GU et al.
2010; EVERTZ et al. 2013; SANCHEZ et al. 2014). Corrosion can be quantified with
different methods like magnesium ion release, weight loss or hydrogen evolution over
time and electrochemical methods or computed tomographical examinations
(WALKER et al. 2014a). For the evaluation of possible toxicity or biocompatibility in
vitro, cell culture studies are used (XU et al. 2009; YANG et al. 2013; SANCHEZ et al.
2014). Beside fibroblasts (WANG et al. 2013; WEIZBAUER et al. 2014), human
osteoblasts (YANG et al. 2013; WEIZBAUER et al. 2014), murine pre-osteoblasts and
mesenchymal stem cells (OSTROWSKI et al. 2013) are used for cytotoxicity studies.
According to the chosen cell type, results can vary already for a single alloy
(WEIZBAUER et al. 2014) and a comparison between different studies is almost
impossible. Cytotoxicity in cell culture studies is often associated with an increase in
osmolality and pH due to the corrosion of the implant material which adds further
interactive variables like solution volume and pH-adjustment to the study design
(WALKER et al. 2014a). In summary, there is no in vitro method, which can predict the
in vivo corrosion characteristics and in vivo biocompatibility until now and in vitro and
in vivo results can differ gravely (WITTE et al. 2006; GU et al. 2009; ZHANG et al.
2010; SANCHEZ et al. 2014). In general, in vivo corrosion rates are lower than in vitro
corrosion rates (WITTE et al. 2006; WALKER et al. 2012; RAHIM et al. 2013;
SANCHEZ et al. 2014; WALKER et al. 2014a).
14
In conclusion, in vitro studies are helpful to perform a first screening of new magnesium
based materials, but need to be evaluated very critical until now. Further work is
necessary to adapt the in vitro systems to the in vivo situation to get more reliable data.
At the present time, in vivo studies are essential to investigate in vivo corrosion and
biocompatibility.
3.2.2. In vivo examination of magnesium based implant materials for orthopedic use
Various different magnesium alloys are already examined in various different animal
models. Most alloys are calcium containing alloys like AX30 (HUEHNERSCHULTE et
al. 2011), MgCa0.8 (THOMANN et al. 2009), MgCa1.0 (LI et al. 2008) or rare earth
containing alloys like WE43 and LAE442 (WITTE et al. 2005; KRAUSE et al. 2010;
WITTE et al. 2010), MgGd (HORT et al. 2009) or LANd442 (own studies). The
aluminum containing alloy AZ91 is additionally tested, but not recommended for in vivo
implantation due to a too fast corrosion rate (WITTE et al. 2005; WITTE et al. 2007c)
and an insufficient mechanical stability under load (GU et al. 2010). In addition, AZ91
has a relatively high aluminum content and toxic effects cannot be excluded (YUEN a.
IP 2010), although WITTE et al. (2007c) did not see any negative effects of the
corroding material on the surrounding bone. In general, no allergic reactions could be
observed for different magnesium alloys (WITTE et al. 2007a) and mild inflammatory
reactions were described as predominantly unspecific (WITTE et al. 2007d).
3.2.2.1. Suitable animal models for the examination of new orthopedic implant
materials
First of all, a clear definition of the particular research question is necessary (PEARCE
et al. 2007). Secondly, the chosen animal model has to reflect the situation, which
15
should be evaluated (DRESPE et al. 2005; PEARCE et al. 2007; MILLS a. SIMPSON
2012). For studies concerning different fracture fixation techniques, rats, rabbits and
sheep are used very commonly (AN a. FRIEDMAN 1998; MARTINI et al. 2001;
REIFENRATH et al. 2014). Additionally, the use of mouse models increased in the last
years, predominantly for the examination of fracture healing on cellular level (HISTING
et al. 2011). Goats, pigs, and dogs are very uncommon animal models in orthopedic
research although the bone microstructure in dogs and pigs is more similar to humans
than in sheep or rabbits (PEARCE et al. 2007). Especially the dog as popular
companion animal is avoided due to ethical aspects. In contrast to mice and rats,
rabbits possess a haversian system similar to larger animals and humans
(MARTINIAKOVÁ et al. 2006).
Special fixation techniques or new implant design are predominately examined in
larger animal models due to a better applicability. For the development of
biodegradable osteosynthesis-systems including novel implant materials intermediate
steps for biocompatibility testing of the materials are indispensable. Different authors
recommend less expensive and complex procedures like subcutaneous, intramuscular
or intravasal implantation in rats or mice prior to intraosseus application (AN a.
FRIEDMAN 1998; MUELLER et al. 2012; WALKER et al. 2012; WALKER et al. 2014b).
However, it has to be taken into account that small animal models like mice and rats
have a faster metabolic rate than larger animals and humans and therewith
degradation rates most probably will differ. Nevertheless, the ease in handling, the
good availability, a huge accessible database and the relatively low costs justify these
models for a lot of experimental designs. The rabbit represents an intermediate animal
model which unites advantages of small animals like relatively unproblematic housing
and handling requirements, a huge amount of evaluation methods as well as a size for
16
the examination of simple implant systems or biomechanical tests. Therewith this
animal model is predominantly used in the following studies.
Nevertheless, the rabbit reaches its limit for the examination of more complex implant
systems like interlocked intramedullary nailing systems. For these implant systems,
the sheep as a large animal model was used. In general, the sheep model is often
used for the study of different bone fixation techniques or materials (REIFENRATH et
al. 2014) or in connection with the development of bone graft materials (BABIKER
2013; CIPITRIA et al. 2013). However, a higher heterogeneity in large animal models
can influence study results more strongly. Therefore, intraindividual comparisons
would be desirable but are limited due to the burden of the animal, which has to be
reduced in accordance to the German animal welfare law, the relevant ethics
committee and the general implementation of the “3R” requirements (RUSSELL a.
BURCH 1992). Additionally handling and costs are increased compared to smaller
animal models. In conclusion, large animal models like sheep are predominately used,
if the research question cannot be answered by the application of a smaller animal
model.
3.2.2.2. Evaluation methods for degradation analysis and biocompatibility in vivo
In the field of degradable magnesium based implant materials, the focus is on the
material´s degradation properties and the biocompatibility. For in vivo degradation
analysis, materials are implanted in animals, left there for a certain time and were
explanted after euthanasia. Weight measurements, radiologic and µ-computed
tomographical evaluation methods can be performed (GU a. ZHENG 2010;
HUEHNERSCHULTE et al. 2011; WALKER et al. 2014a; ZHENG et al. 2014). Weight
analysis prior to implantation and after explantation is a very simple technique to
determine the degradation process over a special period of time. Nevertheless,
17
differences can occur due to variances in the detailed procedure. During the
implantation process, corrosion products and organic material can accumulate at the
implant surface. This has to be taken into account when implants are weighed directly
after explantation (SANCHEZ et al. 2014). To avoid these influences, special
treatments can be performed to detach these products. For magnesium implants,
fluorid- or chromatic acid treatment is a common used method (WALKER et al. 2014a).
After these treatments, corrosion products are removed and only the residual metallic
implant can be weight. However, any adherent tissue has to be removed for this
analysis and further histological examination of the implant interface cannot be
performed. Another disadvantage of weight measurement is the fact that nonlinear
degradation cannot be reliably described. Therefore, other measurement techniques
are advantageous, which can be performed in vivo during the postoperative follow up
period. An often used tool in magnesium research is in vivo µ-computed tomography
(HUEHNERSCHULTE et al. 2011; REMENNIK et al. 2011; WANG et al. 2011; YU et
al. 2012). In contrast to other implant materials like surgical steel or titanium,
magnesium alloys do not cause artifacts during the imaging process. Using this
technique, implant volume, implant density and implant 3D-thickness can be calculated
at different time points over the postoperative follow up and an in vivo degradation
processes can be described (HUEHNERSCHULTE et al. 2011). Therewith, contrary to
the weight measurement method, nonlinear degradation can be indicated. However,
even this method has some weaknesses. In most in vivo µ-computed tomographies,
an insufficient separation of residual implant and adherent corrosion layer has to be
taken into account in the evaluation process. A separation is possible in higher
resolution µ-CT, but these systems are not suitable for the in vivo use and can only be
applied as an additional evaluation tool.
18
Another clinically and radiographically detectable parameter for in vivo degradation of
magnesium implants is the occurrence and accumulation of gas. During the corrosion
process of magnesium, a hydroxid layer is formed and hydrogen is released (SONG
a. ATRENS 2003). In technical applications, the hydrogen evolution is a standard
method for the determination of corrosion (WITTE et al. 2006). In vivo, the emerging
gas dissolves or diffuses in the organism. As soon as these capacities of the organism
are exceeded, gas accumulates and can be radiographically or clinically detected
(WITTE et al. 2005). Therewith in vivo gas detection as an evaluation tool is an
indication for implant degradation, but imprecise. Nevertheless, an exceeding gas
formation should be assessed as undesirable and is described for fast degrading alloys
(WITTE et al. 2005; KRAUS et al. 2012). In conclusion, for exact in vivo degradation
analysis of magnesium implants, a combination of different methods is the gold
standard.
Besides the degradation properties, the biocompatibility is an essential aspect for
implant materials. In orthopedic location, osteoconductive or even osteoinductive
material properties are desired to enhance bone formation and ingrowth of the applied
material. While osteoconductivity is defined as a passive process based on material
parameters which allow the adhesion of bone cells, osteoinductivity is an active
process where cells are attracted and stimulated to differentiate into bone cells
(GRADINGER a. GOLLWITZER 2006). Another aspect of biocompatibility as
orthopedic implant material is the avoidance of specific and unspecific immunological
reactions as well as fibrous encapsulation (AN 2003).
Whereas bone remodeling processes with new bone formation and osteolysis can be
partially evaluated in radiographical and (µ-) computed examinations, which are
described already for the evaluation of the implant degradation, detailed bone
19
remodeling processes on a cellular level, inflammatory reactions and fibrous
encapsulation can only be assessed with histological methods. While in standard
pathological examinations predominantly paraffin embedded specimens are used,
bone tissue, in particular with remaining implant material, is commonly plastic
embedded (AN 2003). In dependence of the research question, the preparation of
microtome slices or grinded slices is advantageous. Whereas slices prepared by the
use of the cutting grinding technique according to DONATH (1995) offer the possibility
to evaluate the direct bone implant interface the slice thickness limits the detailed
evaluation of different cell types. By the use of microtome sections, the residual implant
gets lost during sectioning due to occurring shear forces in the slice, but a higher
variety of histological, histochemical and immunohistochemical staining procedures
can be performed to evaluate the surrounding tissue. Therefore, in accordance to the
special question, both methods are used in the implemented studies.
3.3. Simulation models as an option for reducing animal experiments
When using animal models in research the so-called 3R´s – refinement, replacement
and reduction – (RUSSELL a. BURCH 1992) have always to be taken into account.
Although simulation models cannot predict the true in vivo situation, different
parameters can be examined with computational models. In implant research a stress
adapted bone model might predict implant failure prior to an in vivo animal test and
can avoid unnecessary material failure in the in vivo situation. In the current work,
computed tomographical data of sheep and rabbit tibia as well as experimentally
collected stress data in the rabbit tibia were generated. These data were used as main
elements for the mathematical calculation of tibia models of these two animals for
orthopedic research, which was performed by engineers from the institution of
continuum mechanics, Leibniz University of Hannover.
20
Therefore, the following aspects were part of the present studies:
1. Examination of different magnesium alloys as possible implant materials for
the use in orthopedic location particular with regard to mechanical
characteristics, degradation and biocompatibility in vivo.
2. Influence of handling and storage considering requirements as implant
material.
3. Selection of most suitable materials and examination in application oriented
implant systems (plate-screw-systems and intramedullary nailing systems)
for the use in fracture fixation in weight bearing bones.
4. Identification of biomechanical requirements for fracture fixation in weight
bearing bones with special focus on degradation over time.
5. Comparative assessment of the used animal models for specific research
questions.
21
4. List of Publications (contributing to the current work)
I. Reifenrath, J., Krause, A., Bormann, D., von Rechenberg, B., Windhagen, H.,
Meyer-Lindenberg, A.: Profound differences in biocompatibility of two very similar
Rare-earth containing Mg-alloys, Mat.- Wiss- u.Werkstofftech. 2010, 41, 12, p. 1054–
1061, doi: 10.1002/mawe.201000709
II. Erdmann, N., Angrisani, N., Reifenrath, J., Lucas, A., Thorey, F., Bormann, D.,
Meyer-Lindenberg A.: Biomechanical testing and degradation analysis of MgCa0.8
alloy screws: A comparative in vivo study in rabbits, Acta Biomater., 2010, 7, 3, p.
1421-1428 doi:10.1016/j.actbio.2010.10.03
III. Erdmann, N., Bondarenko, A., Hewicker-Trautwein, M., Angrisani N.,
Reifenrath, J., Lucas, A., Meyer-Lindenberg, A.: Evaluation of the soft tissue
biocompatibility of MgCa0.8 and surgical steel 316L in vivo: a comparative study in
rabbits, Biomed. Eng. Online, 2010, 9, 63
IV. Badar, M., Reifenrath, J., Rittershaus, D., Seitz, J.-M., Bormann, D., Bach, F-
W., Hauser, H., Meyer-Lindenberg, A., Mueller, P.P.: In vitro and in vivo models for
the molecular evaluation of cellular responses to magnesium, Biomed Tech 2010, 55,
Suppl. 1, doi: 10.1515/BMT.2010.125
V. Reifenrath, J., Bormann, D., Meyer-Lindenberg, A.: Magnesium alloys as
promising degradable implant materials in orthopaedic research; Chapter 6 in
Magnesium alloys – corrosion and surface treatments; Czerwinski F, Rijeka Intech,
2011, p. 93-108, ISBN 978-953-307-972-1
VI. Reifenrath, J., Gottschalk, D., Angrisani, N., Besdo, S., Meyer-Lindenberg, A.:
Axial forces and bending moments in the loaded rabbit tibia in vivo; Acta Vet. Scand.
2012, 54, 21, doi:10.1186/1751-0147-54-21
22
VII. Hampp, C., Ullmann, B., Reifenrath, J., Angrisani, N., Dziuba, D., Bormann, D.,
Seitz, J.-M., Meyer-Lindenberg, A.: Research on the Biocompatibility of the New
Magnesium Alloy LANd442 – An In Vivo Study in the Rabbit Tibia over 26 Weeks;
Adv. Eng. Mater. 2011, 14, 3, B28-B37, doi: 10.1002/adem.201180066
VIII. Ullmann, B., Reifenrath, J., Dziuba, D., Seitz, J.-M., Bormann, D., Meyer-
Lindenberg, A.: In Vivo Degradation Behavior of the Magnesium Alloy LANd442 in
Rabbit Tibiae; Materials 2011, 4, p. 2197-218; doi: 10.3390/ma4122197
IX. Huehnerschulte, T. A., Reifenrath, J., von Rechenberg, B., Dziuba, D., Seitz,
J. M., Bormann, D., Windhagen, H., Meyer-Lindenberg A.: In vivo assessment of the
host reactions to the biodegradation of the two novel magnesium alloys ZEK100 and
AX30 in an animal model, Biomed. Eng. Online, 2012, 11, 14
X. Hampp, C., Angrisani, N., Reifenrath, J., Bormann, D., Seitz, J.-M., Meyer-
Lindenberg, A.: Evaluation of the biocompatibility of two magnesium alloys as
degradable implant materials in comparison to titanium as non-resorbable material in
the rabbit, Mater. Sci. Eng. C, 2013, 33, p. 317-26,
doi.org/10.1016/j.msec.2012.08.046
XI. Reifenrath, J., Badar M., Dziuba, D., Müller, P. P., Heidenblut, T., Bondarenko,
A., Meyer-Lindenberg, A.: Evaluation of cellular reactions to magnesium as implant
material in comparison to titanium and to glyconate using the mouse tail model, J.
Appl. Biomater. Funct. Mater., 2013, 11, 2, e89-94, doi: 10.5301/JABFM.5000150.
XII. Ullmann, B., Angrisani, N., Reifenrath, J., Seitz, J.M., Bormann, D., Bach, F.W.,
Meyer-Lindenberg, A.: The effects of handling and storage on magnesium based
implants--first results, Mater. Sci. Eng. C Mater Biol. Appl., 2013, 33, 5, p. 3010-7,
doi: 10.1016/j.msec.2013.03.034.
XIII. Ullmann, B., Reifenrath, J., Seitz, J.-M., Bormann, D., Meyer-Lindenberg, A.:
Influence of the grain size on the in vivo degradation behaviour of the magnesium
23
alloy LAE442, Proc. Inst. Mech. Eng. H J. Eng. Med., 2013, 27, 3, doi
10.1177/0954411912471495
XIV. Bondarenko, A., Angrisani, N., Meyer-Lindenberg, A., Seitz, J.M., Waizy, H.,
Reifenrath, J.: Magnesium-based bone implants: Immunohistochemical analysis of
peri-implant osteogenesis by evaluation of osteopontin and osteocalcin expression.
J Biomed. Mater. Res. A., 2013, 102, 5, p. 1449–57, doi: 10.1002/jbm.a.34828.
XV. Dziuba, D., Meyer-Lindenberg, A., Seitz, J. M., Waizy, H., Angrisani, N.,
Reifenrath, J.: Long-term in vivo degradation behaviour and biocompatibility of the
magnesium alloy ZEK100 for use as biodegradable bone implant; Acta Biomater.,
2013, 9, 10, p. 8548-60, doi.org/10.1016/j.actbio.2012.08.028
XVI. Reifenrath, J., Angrisani, N., Erdmann, N., Lucas, A., Waizy, H., Seitz, J.M.,
Bondarenko, A., Meyer-Lindenberg, A.: Degrading magnesium screws ZEK100:
biomechanical testing, degradation analysis and soft-tissue biocompatibility in a
rabbit model. Biomed. Mater., 2013, 8, 4, p. 045012, doi: 10.1088/1748-
6041/8/4/045012.
XVII. Weizbauer, A., Modrejewski, C., Behrens, S., Klein, H., Helmecke, P., Seitz,
J.M., Windhagen, H., Möhwald, K., Reifenrath, J., Waizy,H.,: Comparative in vitro
study and biomechanical testing of two different magnesium alloys, Biomater. Appl.
J Biomater Appl., 2014, 28, 8, p. 1264-73, doi: 10.1177/0885328213506758.
XVIII. Wolters, L., Angrisani, N., Seitz, J., Helmecke, P., Weizbauer, A.,
Reifenrath J.: Applicability of Degradable Magnesium LAE442 Alloy Plate-Screw-
Systems in a Rabbit Model. Biomed. Tech., 2013, p. 227 doi:pii:
/j/bmte.2013.58.issue-s1-C/bmt-2013-4059/bmt-2013-4059.xml. 10.1515/bmt-2013-
4059.
XIX. Reifenrath, J., Roessig, C., Wolters, L., Seitz, J.-M., Helmecke, P., Angrisani,
N.: Implant location strongly influences degradation and applicability of magnesium
24
alloys for orthopaedic application, Europ. Cells Mat., 2013, 26, Suppl. 5, p.17, ISSN
1473-2262
XX. Reifenrath, J., Angrisani, N., Lalk, M., Besdo, S.: Replacement, refinement and
reduction: necessity of standardization and computational models for long bone
fracture repair in animals, J Biomed. Mater. Res. A., 2014, 102, 8, p. 2884-900
XXI. Rössig, C., Angrisani, N., Besdo, S., Damm, N.B., Badenhop, M., Fedchenko,
N., Helmecke, P., Seitz, J.M., Meyer-Lindenberg, A., Reifenrath, J.: Magnesium-
based intramedullary nailing system in a sheep model: Biomechanic evaluation and
first in vivo results, J. Vet. Sci. Med. Diagn. 2014, 4, 1, doi:10.4172/2325-
9590.1000150
XXII. Bracht, K., Angrisani, N., Seitz, J.M., Eifler, R., Weizbauer, A., Reifenrath, J.:
The influence of storage and heat treatment on a magnesium-based implant material:
an in vitro and in vivo study, Biomed Eng Online. 2015, 14, 92, doi: 10.1186/s12938-
015-0091-8.
XXIII. Wolters, L., Besdo, S., Angrisani, N., Wriggers, P., Hering, B., Seitz, J.M.,
Reifenrath, J.: Degradation behaviour of LAE442-based plate-screw-systems in an
in vitro bone model, J Mat. Sci. Eng. C, 2015, 49, p. 305–15
XXIV. Rössig, C., Angrisani, N., Helmecke, P., Besdo, S., Seitz, J.M., Welke,
B.,Fedchenko, N., Kock, H., Reifenrath, J.: In vivo evaluation of a magnesium-based
degradable intramedullary nailing system in a sheep model, Acta Biomater. 2015, 25,
p. 369-83, doi: 10.1016/j.actbio.2015.07.025 16.03.2015
25
5. Results and Discussion
5.1. In vivo degradation characteristics and biocompatibility of different
magnesium based alloys in contrast to conventional implant materials
I Reifenrath, J., Krause, A., Bormann, D., von Rechenberg, B., Windhagen, H.,
Meyer-Lindenberg, A.: Profound differences in biocompatibility of two very similar
Rare-earth containing Mg-alloys, Mat.- Wiss- u.Werkstofftech. 2010, 41, 12, p. 1054–
1061, doi: 10.1002/mawe.201000709
III. Erdmann, N., Bondarenko, A., Hewicker-Trautwein, M., Angrisani N.,
Reifenrath, J., Lucas, A., Meyer-Lindenberg, A.: Evaluation of the soft tissue
biocompatibility of MgCa0.8 and surgical steel 316L in vivo: a comparative study in
rabbits, Biomed. Eng. Online, 2010, 9, 63
IV. Badar, M., Reifenrath, J., Rittershaus, D., Seitz, J.-M., Bormann, D., Bach, F-
W., Hauser, H., Meyer-Lindenberg, A., Mueller, P.P.: In vitro and in vivo models for the
molecular evaluation of cellular responses to magnesium, Biomed Tech 2010, 55,
Suppl. 1, doi: 10.1515/BMT.2010.125
V. Reifenrath, J., Bormann, D., Meyer-Lindenberg, A.: Magnesium alloys as
promising degradable implant materials in orthopaedic research; Chapter 6 in
Magnesium alloys – corrosion and surface treatments; Czerwinski F, Rijeka Intech,
2011, 93-108, ISBN 978-953-307-972-1
VII. Hampp, C., Ullmann, B., Reifenrath, J., Angrisani, N., Dziuba, D., Bormann, D.,
Seitz, J.-M., Meyer-Lindenberg, A.: Research on the Biocompatibility of the New
Magnesium Alloy LANd442 – An In Vivo Study in the Rabbit Tibia over 26 Weeks; Adv.
Eng. Mater. 2011, 14, 3, B28-B37, doi: 10.1002/adem.201180066
26
VIII. Ullmann, B., Reifenrath, J., Dziuba, D., Seitz, J.-M., Bormann, D., Meyer-
Lindenberg, A.: In Vivo Degradation Behavior of the Magnesium Alloy LANd442 in
Rabbit Tibiae; Materials 2011, 4, p. 2197-218; doi: 10.3390/ma4122197
IX. Huehnerschulte, T. A., Reifenrath, J., von Rechenberg, B., Dziuba, D., Seitz, J.
M., Bormann, D., Windhagen, H., Meyer-Lindenberg A.: In vivo assessment of the host
reactions to the biodegradation of the two novel magnesium alloys ZEK100 and AX30
in an animal model, Biomed. Eng. Online, 2012, 11, 14
X. Hampp, C., Angrisani, N., Reifenrath, J., Bormann, D., Seitz, J.-M., Meyer-
Lindenberg, A.: Evaluation of the biocompatibility of two magnesium alloys as
degradable implant materials in comparison to titanium as non-resorbable material in
the rabbit, Mater. Sci. Eng. C, 2013, 33, p. 317-26,
doi.org/10.1016/j.msec.2012.08.046
XI. Reifenrath, J., Badar M., Dziuba, D., Müller, P. P., Heidenblut, T., Bondarenko,
A., Meyer-Lindenberg, A.: Evaluation of cellular reactions to magnesium as implant
material in comparison to titanium and to glyconate using the mouse tail model, J. Appl.
Biomater. Funct. Mater., 2013, 11, 2, e89-94, doi: 10.5301/JABFM.5000150.
XIII. Ullmann, B., Reifenrath, J., Seitz, J.-M., Bormann, D., Meyer-Lindenberg, A.:
Influence of the grain size on the in vivo degradation behaviour of the magnesium alloy
LAE442, Proc. Inst. Mech. Eng. H J. Eng. Med., 2013, 27, 3, doi
10.1177/0954411912471495
XIV. Bondarenko, A., Angrisani, N., Meyer-Lindenberg, A., Seitz, J.M., Waizy, H.,
Reifenrath, J.: Magnesium-based bone implants: Immunohistochemical analysis of
peri-implant osteogenesis by evaluation of osteopontin and osteocalcin expression. J
Biomed. Mater. Res. A., 2013, 102, 5, p. 1449–57, doi: 10.1002/jbm.a.34828.
27
XV. Dziuba, D., Meyer-Lindenberg, A., Seitz, J. M., Waizy, H., Angrisani, N.,
Reifenrath, J.: Long-term in vivo degradation behaviour and biocompatibility of the
magnesium alloy ZEK100 for use as biodegradable bone implant; Acta Biomater.,
2013, 9, 10, p. 8548-60, doi.org/10.1016/j.actbio.2012.08.028
In the current studies, magnesium itself and different alloying materials (LAE442,
LANd442, LACer442, WE43, ZEK100, AX30, MgCa0.8) were predominantly examined
with regard to degradation properties and biocompatibility in vivo. Whereas LAE442
contains 4 wt% lithium, 4 wt% aluminum and 2 wt% rare earths adjacent to magnesium,
in the alloys LACer442 and LANd442 the rare earth mixture was replaced by a
specified single rare earth element (cerium or neodymium) to ensure a better
reproducibility of the bulk material. MgCa0.8 was chosen for further studies because
previous examinations showed good biocompatibility in osseous location (KRAUSE
2008). The alloys AX30 and ZEK100 were new developed alloying materials with
adequate mechanical characteristics for the later use as orthopedic implant material.
Biocompatibility studies of the different Mg-based alloys were predominantly
performed in rabbit tibiae (studies I, V, VII, VIII, IX, X, XI, XIII, XIV, XV). The used
model was well known from former studies (KRAUSE et al. 2005; THOMANN et al.
2009; HUEHNERSCHULTE 2009), which were carried out in our research group.
Therewith, comparisons to already examined Mg-based alloys and common used
permanent (titanium) and degradable (PLA) implant materials were possible (KRAUSE
2008). The basic principle was to implant Mg-based pins (2.5 mm in diameter, 25 mm
length) intramedullary in the rabbit tibia. Therefore, the rabbits were anesthetized and
the operation field was clipped and disinfected. After a skin incision on the medial tibia
plateau, a hole (Ø 2.5 mm) was drilled for implant insertion. For correct placement in
the medullary cavity, the pin was pushed inside with a plastic stick. Wound closure was
28
performed layer by layer (periosteum/ fascia and skin) with resorbable suture material.
The implant location was radiographically verified in two planes immediately after
surgery. Degradation and biocompatibility were examined by the use of clinical,
radiographical, µ-computed tomographical and histological methods as well as, in
some studies, by additional weight analyses, three-point bending tests or scanning
electron microscopy-examinations of the residual implant material after euthanasia.
Clinical examinations gave first evidences, if gas development during implant
degradation exceeded the capacity of the organism to diffuse or resorb it and therewith
might cause clinical problems. Whereas in the very fast degrading alloy LACer442 gas
formation was clinically visible after two weeks and clinical problems like lameness and
pain occurred (study I) in slower degrading alloys like LAE442, LANd442, AX30 and
ZEK100 (studies I, X, XI, XII, XIII) as well as MgCa0.8 (THOMANN et al. 2010b)
lameness occurred only in one animal in the LANd442 group and only very few animals
showed a palpable emphysema or gas bubbles under the skin. Other authors found
gas formation around intramedullary implanted magnesium based AZ91, AZ31, WE43
and LAE442 alloys in guinea pigs (WITTE et al. 2005) and around AZ31 based
orthopedic screws in hip bone of sheep (WILLBOLD et al. 2011) which disappeared
two to three weeks postoperatively and did not cause clinical problems like lameness
or visible pain. More noticeable amounts of gas could be clinically detected in
implanted magnesium based screws (MgCa0.8, ZEK100) which were in contact to the
overlaying soft tissue and induced gas bubbles adjacent to the screw head directly
under the skin (studies III and XVI). However, in most cases, occurring gas cavities did
not affect the animals.
For a more detailed evaluation of implant effects and ongoing degradation in vivo,
different imaging techniques were used, which also helped to assess the formation of
29
gas more precisely. In radiological images, smaller amounts of gas can be detected
than in the clinical examination. Very slow degrading alloys like LAE442 did not show
radiographically visible gas formation during the implant degradation in contrast to fast
degrading alloys like LACer442. For the intramedullary implanted alloys ZEK100 and
AX30 small amounts of gas could be observed in the radiographic examination only in
few animals at the later time points 20 and 24 weeks postoperatively
(HUEHNERSCHULTE et al. 2011). In contrast, ZEK100 alloys in soft tissue contact,
when implanted as bone screws, induced gas pouches under the skin (Fig. 1). Other
authors found gas pouches near the implanted material in Mg-Zn alloys in the first three
weeks of implantation time (ZHANG et al. 2010).
Fig. 1: Fast degrading alloys like LACer442 induced gas pouches under the skin in contrast to slower
degrading alloys like ZEK100 and LAE442 when implanted intramedullary. In contrast to intramedullary
implantation, gas formation was visible in ZEK100 screws in soft tissue contact.
For semiquantitative analysis of radiographic pictures, scoring systems were used with
special focus on bone growth at the implant location and diaphysis, changes in the
medullary cavity and the cortex as well as gas formation. Whereas in some of the
studies (study X and XIII) total scores were used to compare different alloys, in other
30
studies the different parameters were evaluated as single data (study I). Both methods
have advantages and disadvantages. Whereas a differentiation between single
parameters is impossible in total scores, total impact of different materials is presented
more clearly. For the faster degrading alloys, especially LaCer442, massive bone
reactions like periosteal proliferation and osteolysis could be observed (study I). In
other studies the extent of changes was much lower. For LAE442 and LANd442, which
was compared to titanium and a control group without implant material (study X), the
difference in total score after 8 weeks was only 1 score point (maximum value 15
points). However, in this study the postoperative observation time was only 8 weeks
and in slow degrading alloys, bone alterations may still occur at a later time point.
A more detailed but time-consuming and cost-intensive method is µ-computed
tomography. This technique provides information about changes in implant volume and
density as well as bone reactions in the direct implant surrounding. It is used by a large
number of investigators (WONG et al. 2010; REMENNIK et al. 2011; YU et al. 2012),
well established in our own research group (KRAUSE et al. 2010;
HUEHNERSCHULTE et al. 2011) and a major evaluation tool in most of the
implemented studies. Two different µ-computer tomographs were used; the µCT80
and the XtremeCT (both Scanco medical, Switzerland). Whereas in the µCT80 a higher
resolution of up to 10µm can be achieved the XtremeCT is limited to 41µm. However,
in the XtremeCT in vivo measurements can be performed, which is not possible in the
µCT80 and, therewith, only a final evaluation at the end of the postoperative
observation time can be done. For that reason most studies implemented both
techniques with different focuses concerning the study question. Implant degradation
was evaluated either in selected cross sections (study I, V or with the help of specific
software (V6.1, Scanco Medical, Zürich, Switzerland) to determine loss of implant
volume (Fig. 2) and density as well as bone volume and porosity, respectively.
31
Fig. 2: Exemplary depiction of measured volume losses of different pin materials implanted in rabbit
tibiae by the use of in vivo µ-computed tomography.
On the basis of volume losses, corrosion rates can be calculated according to the
following formula:
𝐶𝑅 = 365 ∗ ∆𝑉/(𝐴 ∗ 𝑡)
with CR [mm/year] is the corrosion rate, ΔV [mm3] the volume loss, A [mm2] the surface
which was subjected to the corrosion and t [days] the implantation period (WITTE et
al. 2010). For the different tested alloys various different corrosion rates were
calculated. For LANd442corrosion rates between 0.01 mm/y and 0.072 mm/year were
observed, depending on the observation time. For LAE442 corrosion rates between
0.03 and 0.04mm/y (48 weeks implantation time, untreated and heat treated materials,
respectively; study XXII) can be calculated from volume losses measured by in vivo µ-
computed tomography. HUEHNERSCHULTE et al. (2011) calculated for ZEK100 and
AX30 corrosion rates of 0.065 mm/y and 0.11 mm/y in the AX30 3 months and 6
32
months groups, respectively. For ZEK100 alloys 0.067 mm/y and 0.154 mm/y in the 3
and 6 months groups are described. These results show that calculated corrosion rates
strongly depend from the investigation time; because magnesium alloys did not show
a linear corrosion process and results cannot be compared directly.
Besides differences in bulk material, corrosion depends on the implant structure and
manufacturing process. For LAE442 implants (study XIII), different corrosion rates for
LAE442 were observed depending on different grain sizes due to the fabrication
process (with and without additional extrusion protocols after die casting) and
additional artificial surface defects. The highest corrosion rate after 2 weeks was
determined for implants with defects (0.121mm/year). At the end of the observation
period of 27 weeks, two times extruded implants with the finest grain size exhibited the
lowest corrosion rate (0.013 mm/year) compared not extruded and single extruded
implants after the die casting process with 0.035 and 0.025 mm/year, respectively,
which was comparable to the calculated corrosion rates for LAE442 in study XXII (48
weeks implantation time) with 0.03 mm/y. The implants with defects again showed the
highest corrosion rate with 0.04mm/year at this time point. Therewith calculated
corrosion rates for LAE442 in our studies were about 10 fold lower than calculated
corrosion rates by WITTE et al. (2010). One explanation might be the different study
design. Whereas WITTE et al implanted the LAE442 alloys in trabecular femoral bone,
in our studies the pins were implanted intramedullary in the rabbit tibiae. Other
explanations are differences in used volume measurements. Although in both cases
µ-computed tomography was used, the resolution in our studies was lower than in the
synchrotron radiation based µ-computed tomography used by WITTE et al. (2010).
Therewith, they were able to distinguish corrosion products e.g. calcium precipitates at
the implant surface as well as small pits from the residual implant material, which was
not possible in our in vivo µCT studies. In conclusion, the measured volume in in vivo
33
µ-CT and the subsequent calculated corrosion rate can be lower than the actual
volume and corrosion rate. These assumptions can be verified with additional weight
measurements after removal of corrosion products by the use of chromatic or fluoric
acid. In study XIII, e.g. after 26 weeks a 5.5% volume loss was measured in in vivo
µCT and a weight loss of 14% after treatment with chromatic acid. These results
suggest that comparisons between different methods and studies remain difficult, and
methodological errors always have to be taken into account when results are
interpreted. However it can be summarized that LAE442 and LANd442 degrade
significantly slower than ZEK100 and AX30 implant materials.
For further characterization of the explanted residual implant material at the end of the
postoperative follow up period, scanning electron microscopy was used. Fig. 3 shows
exemplary pictures of different alloying materials after 3 and 9 months implantation
time compared to an unaltered implant prior to implantation.
Comparable evaluation of mechanical strength in three-point-bending tests and weight
loss during implantation of different alloying materials is shown in Fig. 4.
34
Fig. 3: Used alloying materials show differences in corrosion progress; whereas in LACer442 alloys
deep pits are observable, LANd442 and ZEK100 surfaces are more homogeneous after 3 months
implantation time. After 9 months, ongoing degradation is more prominent in ZEK100 compared to
LAE442 alloys.
Fig. 4: Weight loss (a) and mechanical stability (b) of the different implant materials after three and six
(all materials) and twelve months (LAE442 and MgCa0.8); Published in: Reifenrath, J., Meyer-Lindenberg, A.
Magnesium Alloys as Promising Degradable Implant Materials in Orthopaedic Research in: Magnesium Alloys - Corrosion and
Surface Treatments ISBN 978-953-307-972-1, 2011
Considerable faster degrading alloys were tested in vivo by KRAUS et al. (2012), who
implanted ZX50 alloys in rat femora and caused excessive changes in the surrounding
bone. They stated that the gas pressure due to large amounts of hydrogen gas during
the fast corrosion process induced some mechanical disturbance of bone
Weight loss of implant materials
0,06
0,09
0,12
0,15
0,18
0,21
0,24
0 3 6 12
time in months
we
igh
t in
g LAE442
WE43
MgCa0.8
AX30
ZEX100
Three point bending test
0
50
100
150
200
250
300
0 3 6 12
time in months
Fm
ax
in
N
LAE442
WE43
MgCa0.8
AX30
ZEX100
a b
35
regeneration. In µ-computed tomographical analyses they saw large gas bubbles and
calculated a daily formation of ~270mm3 H2 in conjunction with almost complete pin
degradation after 12 weeks. Although bone alterations were very severe, KRAUS et
al. (2012) stated a restitution ad integrum recovery of the bone approximately 12 weeks
after the complete degradation of the implanted material and no residual inflammatory
signs.
Nevertheless, observed bone reactions in their study are not acceptable for the clinical
use. Therefore, a slow degradation rate is mandatory in osseous environment because
gas cannot disperse as it is possible in soft tissue, vessels or ventilated applications
like intranasal stents. For the slower degrading implant materials we used in the
subsequent studies (studies V, VII, IX, X), different degrees of bone reactions could be
observed. In general it could be stated that the corrosion rate seemed to influence
bone remodeling to a higher extend than different alloying elements. A slower
degradation induced fewer changes in bone structure than a faster degradation (study
V).
For a more detailed evaluation, beside the µ-computed analysis of changes in bone
structure, cellular responses are of utmost importance. For bone remodeling
properties, osteoclasts and osteoblast are the most important cells (PARFITT 1994).
Additionally, in the assessment of implant biocompatibility, inflammatory reactions and
tissue damage should not be neglected. Therefore, histological examinations at the
end of the postoperative observation period should be performed as far as possible in
the direct implant surrounding. For the evaluation of systemic toxicity, the investigation
of excretory organs and inflammatory indicators in blood samples are feasible tools. In
the present studies, organ samples (liver, spleen, kidney) were examined after
implantation periods of up to one year for the alloy ZEK100. Interleukin-6 as systemic
36
indicator for inflammation (TANAKA a. KISHIMOTO 2014) was analyzed after 6
months implantation period of LANd442 pins. Pathological changes could not be
observed in any paraffin embedded and H&E stained sections (study XV) and
interleukin-6 levels were unchanged (study VII). These exemplary analyses indicated
no systemic toxicity of magnesium based implants although longer time periods should
be examined. Additionally, organ samples should be carefully analyzed when implants
with a larger geometry and therewith an increased amount of total elements are used.
The concentration of alloying elements in these samples by the use of refined analytical
methods like inductive coupled plasma mass spectroscopy is an objective for further
studies.
In contrast to absent systemic influences by the implants, local effects were detectable
in various peculiarities. In most cases, bone remodeling processes were generally
increased around magnesium implants. Additionally it can be stated that bone
remodeling activities depend on the implant degradation rate. Whereas slow degrading
alloys like LAE442 predominantly show only few cavities in the cortical bone, in faster
degrading alloys like AX30 periosteal and endosteal reactions were increased.
Exemplary pictures are shown in Fig. 5.
Fig. 5: Exemplary toluidine-blue stained histological sections of bones with different magnesium alloys.
In AX3-alloys, periosteal and endosteal bone reactions as well as bone cavities in the cortical bone are
37
more pronounced than in the other alloys LANd442 and LAE442. Bone ongrowth at the implant surface
can be observed in the LAE442 alloy with an implantation period of 9 months.
Other authors also described increased bone remodeling activities in conjunction with
magnesium based implants in various studies (ZREIQAT et al. 2002; REVELL et al.
2004; WITTE et al. 2005; WILLBOLD et al. 2013).
In comparison to common used non degradable and degradable materials like surgical
steel and titanium as well as polyglycolid acid, respectively, inflammatory reactions and
fibrous encapsulation were reduced around magnesium implants (studies X, XI).
During the corrosion process of magnesium in vivo, calcium and phosphate
precipitates could be found at the implant surface already after two weeks. Similar
results were found in synthetic media in vitro (KUWAHARA et al. 2000). These calcium
phosphate precipitates at the implant surface might cause minor inflammatory
reactions compared to the conventional used materials.
Similar reduced inflammatory soft tissue reactions were also found in study III, where
soft tissue in direct contact to implanted bone screws was examined (MgCa0.8 alloy
versus surgical steel). These findings were confirmed in in vitro studies with
macrophages, where magnesium corrosion particles showed a low inflammatory and
immunogenic potential (ROTH et al. 2014). Other authors, however, saw no
differences in fibrous encapsulation and the appearance of inflammatory cells like
lymphocytes and macrophages between intramuscular implanted Mg–0.4Ca, Mg–
0.8Ca, Mg–0.5Mn and Mg–1Zn alloys compared to titanium (WALKER et al. 2014b).
In conclusion, for magnesium and its alloys in general, a good biocompatibility can be
stated.
The first studies in osseous environment indicated an osteoinductive effect of
magnesium based alloys (study V). This effect is assumed by other authors as well
38
(ZREIQAT et al. 2002; REVELL et al. 2004) but had not been clearly demonstrated at
that time point. Osteoinduction is defined as a process by which osteogenesis is
induced regardless of osseous environment. The recruitment of immature cells and the
stimulation of these cells to develop into preosteoblasts are essential for osteoinductive
properties (ALBREKTSSON a. JOHANSSON 2001). In orthotopic location, it is difficult
to distinguish between osteoinduction and osteoconduction, which defines bone
growth on an implant surface. Therewith these two definitions were not always clearly
separated. As an osteoinductive effect would be very desirable in fracture repair, even
for defects with the risk of a non-union, it was the aim of study XI to evaluate the
osteoinductive potential of magnesium in comparison to glyconate and titanium as
degradable and nondegradable common used materials in an ectopic location. The
mouse tail model was chosen, which was established in the research group (study IV).
Magnesium, glyconate and titanium wires were implanted in tail veins of mice. After
different time intervals (2, 4, 8 weeks for all materials and additionally 16 and 32 weeks
for magnesium as well as 24 weeks for glyconate and titanium) µ-computed
tomography, histology and EDX-examinations were performed to examine bone
formation and inflammatory reactions as well as the degradation in the magnesium and
glyconate groups. Whereas calcium phosphate precipitates were observed around
magnesium implants already after 2 weeks, chondromatosis or cellular bone structures
could not be found even after 32 weeks observation period although HABIBOVIC and
DE GROOT (2007) assumed that calcium and phosphorous might act as physic-
chemical trigger for local stem cells to differentiate into the osteogenic lineage. Even if
the desired osteoinductive effects of magnesium could not be proved, calcium
phosphate precipitates on the surface of orthopedic implants are assessed as a
positive factor for new bone formation and therewith induce osteoconductive effects.
Therefore, the first hypothesis that magnesium has osteoinductive properties had to
39
be revised. Nevertheless, for orthopedic implant materials even an osteoconductive
property, which can be stated for the magnesium alloys, is a favorable characteristic.
In bone-implant interactions and bone remodeling processes, different regulatory
pathways are involved. Many biological substances are known, which up- or down
regulate bone growth, bone healing and bone remodeling (GUNDBERG 2003). Two of
these matrix proteins, osteocalcin and osteopontin, were analyzed in study XIV around
MgCa0.8, LAE442, LANd442 and ZEK100 implants compared to titanium after 3 and
6 months implantation time. For all implants, an increase in osteocalcin expression was
associated with an increased level of new bone formation. Decreasing corrosion rates
of LAE442 as well as increasing corrosion rates of MgCa0.8 were virtually in line with
osteocalcin expressions. Whereas osteocalcin regulates the bone mineralization,
osteopontin provides osteoclast migration and adhesion. The evaluated osteopontin
expressions correlated less with bone forming/ remodeling properties. Osteopontin is
mainly expressed in the early stage of bone healing and has a predominantly adhesive
function as a proinflammatory mediator and attracts cells, especially osteoclasts, to the
site of injury (MCKEE et al. 2011). Whereas increased osteopontin levels in ZEK100
and LANd442 implants after 6 months implantation time might have been caused by
inflammatory processes, increased levels in the titanium group after 3 months
implantation time were presumably induced by pronounced osteoinductive properties
which are also mentioned by other authors (DEPPRICH et al. 2008). In conclusion, the
examined bone markers are first steps to understand the bone implant interactions in
magnesium alloys deeper and declining levels of osteopontin and osteocalcin over
time indicated a good biocompatibility, especially for LAE442. However, further
examination on regulatory pathways and matrix proteins are necessary to explain
implant tissue interactions in magnesium based materials.
40
Summarized, the most favorable alloying materials concerning biocompatibility were
slow degrading alloys like LANd442, ZEK100 and LAE442; especially LAE442 differed
from all others concerning very slow degradation rate and good biocompatibility with
bone ongrowth at the implant surface and decreased endosteal and periosteal bone
remodeling properties compared to faster degrading alloys.
5.2. Influence of handling and storage on magnesium based implants
XII. Ullmann, B., Reifenrath, J., Seitz, J.-M., Bormann, D., Meyer-Lindenberg, A.:
Influence of the grain size on the in vivo degradation behaviour of the magnesium alloy
LAE442, Proc. Inst. Mech. Eng. H J. Eng. Med., 2013, 27, 3, doi
10.1177/0954411912471495
XXII. Bracht, K., Angrisani, N., Seitz, J.M., Eifler, R., Weizbauer, A., Reifenrath, J.:
The influence of storage and heat treatment on a magnesium-based implant material:
an in vitro and in vivo study, Biomed Eng Online. 2015, 14, 92, doi: 10.1186/s12938-
015-0091-8.
For the use of implant materials in orthopedic surgery it is indispensable to ensure
constant material properties over a defined period of time. In study XII, a change in the
microstructure (levels of precipitations), the grain size and an increase in oxygen rich
layers at the implant surface was indicated during the course of the implants´ storage.
However, only a low number of pins were examined (only one exemplarily analyzed
pin after each storage period) and it could not be excluded that differences might have
been caused by incidental differences in grain sizes in the original materials. Other
authors found changes in biomechanical characteristics after storage periods up to
1.25 years at room temperature (KOMATSU et al. 2004) with a slow increase in
41
resistivity for ZK60 and MgZn (4–10mass% Zn) alloys in dependence to the aging
duration. To improve mechanical characteristics in a shorter time period, artificial aging
is described, which is triggered by the use of temperatures higher than 100°C over a
certain period of time (BUHA 2008; HE et al. 2010). This procedure can even cause
structural changes e.g. precipitates at grain boundaries (LI et al. 2011). For an increase
in oxygen rich layers at the implant surface, which was observed in study XII during
prolonged storage, it is assumed that corrosion resistance increases. Therefore the
hypothesis for study XXIII was that storage as well as heat treatment influence the
implant characteristics and decrease the corrosion rate in LAE442 pins. However, this
hypothesis could not be confirmed for dry storage at room temperature. Although,
similar to study XII, an increase in oxygen rich layers at the implant surface could be
observed during storage periods up to 48 months, no significant difference in corrosion
rates could be observed in in vitro corrosion testing or in in vivo implantation in rabbit
tibia with a 1 year postoperative follow up between the groups with different storage
periods (0, 12, 24, 48 weeks). In contrast, a decreased corrosion rate and more filiform
corrosion type were approved for implants which were heat treated (180°C, 2h) prior
to corrosion testing. Similar findings could be observed in an in vitro study, where heat
treated AZ63 alloys were compared to untreated samples. In this study, a change in
grain structure and precipitations with corresponding reduced in vitro corrosion rate
could be found in heat treated samples as well as shallow filiform and pitting corrosion
characteristics compared to deep and uniform corrosion characteristics in the
untreated samples (LIU et al. 2007). Therewith, heat treatment might be a possible
alternative to reduce corrosion rate in Mg-alloys. However, for LAE442 alloys, the initial
stability was decreased after the used heat treatment process in study XXIII, which is
in contrast to another study where MgYZ-alloys were aged at 200°C and showed an
increase in tensile strength. Therefore, it must be carefully considered prior to an in
42
vivo application of these implant materials, if mechanical strength or uniform corrosion
is the more important implant characteristic, depending from the target application. The
influence of dry storage up to one year at room temperature on the implant material
can be assessed as negligible, which is an important conclusion for the use of
magnesium based implant materials in clinical applications.
5.3. Application oriented complex mg-based implant materials (plate-screw-
systems and intramedullary nailing systems) for fracture fixation in weight
bearing bones
II. Erdmann, N., Angrisani, N., Reifenrath, J., Lucas, A., Thorey, F., Bormann, D.,
Meyer-Lindenberg A.: Biomechanical testing and degradation analysis of MgCa0.8
alloy screws: A comparative in vivo study in rabbits, Acta Biomater., 2010, 7, 3, p.
1421-1428, doi:10.1016/j.actbio.2010.10.03
XVI. Reifenrath, J., Angrisani, N., Erdmann, N., Lucas, A., Waizy, H., Seitz, J.M.,
Bondarenko, A., Meyer-Lindenberg, A.: Degrading magnesium screws ZEK100:
biomechanical testing, degradation analysis and soft-tissue biocompatibility in a rabbit
model. Biomed. Mater., 2013, 8, 4, p. 045012, doi: 10.1088/1748-6041/8/4/045012.
XVII. Weizbauer, A., Modrejewski, C., Behrens, S., Klein, H., Helmecke, P., Seitz,
J.M., Windhagen, H., Möhwald, K., Reifenrath, J., Waizy,H.,: Comparative in vitro
study and biomechanical testing of two different magnesium alloys, Biomater. Appl. J
Biomater Appl., 2014, 28, 8, p. 1264-73, doi: 10.1177/0885328213506758.
XVIII. Wolters, L., Angrisani, N., Seitz, J., Helmecke, P., Weizbauer, A., Reifenrath J.:
Applicability of Degradable Magnesium LAE442 Alloy Plate-Screw-Systems in a
43
Rabbit Model. Biomed. Tech., 2013, p. 227 doi:pii: /j/bmte.2013.58.issue-s1-C/bmt-
2013-4059/bmt-2013-4059.xml. 10.1515/bmt-2013-4059.
XIX. Reifenrath, J., Roessig, C., Wolters, L., Seitz, J.-M., Helmecke, P., Angrisani,
N.: Implant location strongly influences degradation and applicability of magnesium
alloys for orthopaedic application, Europ. Cells Mat., 2013, 26, Suppl. 5, p.17, ISSN
1473-2262
XXI. Rössig, C., Angrisani, N., Besdo, S., Damm, N.B., Badenhop, M., Fedchenko,
N.,Helmecke, P., Seitz, J.M., Meyer-Lindenberg, A., Reifenrath, J.: Magnesium-based
intramedullary nailing system in a sheep model: Biomechanic evaluation and first in
vivo results, J. Vet. Sci. Med. Diagn. 2014, 4, 1, doi:10.4172/2325-9590.1000150
XXIII. Wolters, L., Besdo, S., Angrisani, N., Wriggers, P., Hering, B., Seitz, J.M.,
Reifenrath, J.: Degradation behaviour of LAE442-based plate-screw-systems in an in
vitro bone model, J Mat. Sci. Eng. C, 2015, 49, p. 305–315
XXIV. Rössig, C., Angrisani, N., Helmecke, P., Besdo, S., Seitz, J.M., Welke, B.,
Fedchenko, N., Kock, H., Reifenrath, J.: In vivo evaluation of a magnesium-based
degradable intramedullary nailing system in a sheep model, Acta Biomater. 2015, 25,
p. 369-83, doi: 10.1016/j.actbio.2015.07.025 16.03.2015
After the general examination for biocompatibility in orthotopic location in small animal
models, Mg-based implant materials were examined in application oriented studies for
the use as orthopedic implant materials. Therefore simple orthopedic implant
geometries like screws and small plate-screw-systems were implanted in rabbits as
small animal model and a more complex interlocked nailing system in sheep as large
animal model (Fig. 6).
44
Fig. 6: Different used orthopedic implant geometries for further investigations
Only the Mg-alloys MgCa0.8, ZEK100 and LAE442, which were evaluated as
promising biodegradable materials in former studies, were chosen for further
investigations. Beside a good biocompatibility, ZEK100 and LAE442 excelled with a
very high mechanical strength. In the studies II and XVI, MgCa0.8 screws and ZEK100
screws were implanted in rabbit tibia and functional tests were performed after 2, 4, 6
and 8 weeks postoperative follow up periods. These tests should provide the central
information if the holding power of the screws in the bone was comparable with
conventional used materials like surgical steel and how the degradation influenced the
holding power in the time period of assumed fracture healing. A uniaxial pull-out test
was used to measure pull-out forces in a load displacement curve until failure of bone
or screw. Although the initial bending strength of ZEK100 pins was higher than that of
MgCa0.8 pins (study V), the screw retention forces of ZEK100 screws were slightly
lower after 4 and considerably lower after 6 weeks implantation time in comparison to
MgCa0.8 screws. While the proceeding degradation of Mg-based screws which could
be shown in µ-computed tomography measurements caused a decrease in holding
power over time, the retention forces continuously increased in the control group with
surgical steel screws. However, during secondary fracture healing, callus formation
reduces the interfragmentary movement (CLAES a. HEIGELE 1999) and the
biomechanical load will be reduced over time. Nevertheless, it is questionable, whether
approximately 50% reduction in screw retention forces after six weeks in ZEK100
45
screws are sufficient for their use as osteosynthesis material. A slightly slower loss in
holding power, which could be observed in MgCa0.8 screws, seemed to be more
promising although even for MgCa0.8 a final statement could not be given.
Parallel to the in vivo experiments with Mg-based screws, in vitro examinations of
osteosynthesis plates were performed with the same alloying materials (MgCa0.8 and
ZEK100). Therefore, immersion tests were performed in Hank´s Balanced Salt
Solution at 37°C for a time period of 96h. Four point bending test was used to
determine the initial strength compared to the strength after in vitro corrosion. In both
plate types, a loss in strength of approximately 7% could be observed with a more
prominent pitting corrosion in the MgCa0.8 plates and an 11% lower initial bending
strength. Due to these findings, the ZEK100 alloy was assessed as more suitable for
the use in osteosynthesis systems. However, long term studies of ZEK100 showed an
insufficient biocompatibility in orthotopic location (intramedullary cavity of rabbit tibiae)
for theses alloys with severe bone alterations although having no negative clinical
effects on the animals. In µ-computed tomographical and histological examinations
after implantation times of up to 12 months, a decrease in bone density and an increase
in the number of osteoclasts could be observed (study XV). Therefor this alloy was
excluded for further investigations. In order to combine slow degradation rate, good
biocompatibility and high mechanical strength, the alloy LAE442 was chosen for further
investigations of osteosynthesis systems.
Osteosynthesis plates were examined in the rabbit model in vivo (study XVIII) and
corresponding in vitro corrosion experiments were performed (study XXIII). Although
in former studies a very good biocompatibility of this alloying system was stated, in vivo
results of implanted plate-screw-systems in a preliminary study showed contrary
results. High amounts of gas which caused clinical lameness was observed. In µ-
46
computed tomography and histology examinations, severe periosteal reactions were
found predominantly around the edges of the plates. In areas of direct contact between
plates and underlying bone osteolysis could be observed. Control groups of surgical
steel, in contrast, showed no osteolysis processes and periosteal reactions to a lower
content. An increased bone ingrowth of magnesium based plates could be observed
by other authors as well. CHAYA et al. (2014) examined pure magnesium plates-
screw-systems for low loaded application in a rabbit osteotomy model (ulna) and found
an increase in overlaying bone formation predominantly between 8 and 16 weeks
postoperatively. However, they did not see osteolytic bone reactions and did not
describe clinical relevant gas formation. Therewith, they assessed the material as
potential fixation device. Nevertheless, pure magnesium could not be an alternative for
the use in load bearing applications due to the insufficient mechanical strength. A
possible approach to reduce corrosion rate is the application of coatings. In study XXIII,
the in vivo used osteosynthesis-systems were examined in an in vitro setup, most
possible adapted to the in vivo situation: Plate-screw systems were fixed on explanted
rabbit tibiae and corroded for 14 days in a temperature and pH controlled simulated
body fluid. Different screw torques (7cNm and 15cNm), as well as different
pretreatments (NaOH pretreatment and additional fluoride coating) were used and the
corrosion rate was determined by hydrogen evolution, weight loss and volume loss.
While fluoride coating induced a significant reduction in corrosion rate, NaOH
pretreatment did not have a corrosion protective effect. The effect of fluoride coating
on Mg-based alloys is described oppositely in the literature. Whereas some authors
found only slight corrosion protective effects (THOMANN et al. 2010a) others could
see a significantly reduced corrosion rate in fluoride coated implants (WITTE et al.
2010). Therewith, fluoride coating of plate-screw systems might be an alternative to
reduce corrosion rate and to increase biocompatibility in vivo.
47
In contrast to the LAE442-based osteosynthesis plates in the rabbit model, interlocked
intramedullary nails in the sheep model (studies XXI, XXIV) did not show clinical
problems due to fast corrosion and emerging gas, although the overall amount of
implanted material was even higher compared to the plates. Some gas was visible in
radiographic pictures in the areas of the screw heads, partially in the knee joint after
surgery and in the medullary cavity. However, the animals were not clinically influenced
by this.
Beside the detection of gas formation, computed tomography was used to calculate
changes in implants volume and density during the postoperative follow-up period of
24 weeks. Whereas in the computed tomography the decrease in volume of the
measured nail and two of the four interlocking screws was not significant, the density
even showed a slight increase, which was even significant for screw 1 (study XXIV).
An increase in screw density has been observed in the pilot study as well (study XXI),
although there, the increase concerned screw four instead of screw one combined with
a measured volume loss of approximately 4-6% in both screws. But even in the nails
and screws without detectable computed tomographical volume loss, degradation took
place over time. Indicators for ongoing degradation were detectable gas as well as a
reduced stiffness of the nail after explantation. Whereas stiffness in steel nails
remained almost unaffected as expected (decrease 3.44 %, p=0.186), magnesium
nails showed a significant decrease in stiffness of approximately 25% (25.17 %,
p=0.008). The corresponding determined 0.2 yield point, which defines the stress at
which a material begins to deform plastically, showed a 12.75 % decrease (p=0.008)
compared to the initial value of unimplanted nails after 24 weeks (study XXIV).
Therewith degradation over time could be stated and the material might be appropriate
to decrease stress shielding effects in orthopedic fracture repair.
48
No measurable significant changes in volume loss combined with a significant
decrease in maximum force in the three point bending tests were seen for 26 weeks
implanted LANd442 pins in rabbit tibiae as well (study XIII). Possible causes for these
results were the tending of magnesium for pitting corrosion and a reduction in the
implant diameter, which cannot be detected in computed tomography but which
weakens the material. Additionally, corrosion products are included in the CT-data
based volume calculation, because in computed tomography analysis a differentiation
between residual implant material and corrosion products is often not possible. For a
more detailed evaluation, parts of the implant bone compound were scanned
additionally by µ-computer tomography, with a resolution of 41 µm. Parts of the residual
nail as well as exemplary screws were additionally scanned after detachment of
corrosion products by chromatic acid treatment. Pitting corrosion at the implant surface
could be stated with these methods, which underline ongoing degradation, although
not detectable in computed tomography.
Different other authors found pitting corrosion in in vivo (THOMANN et al. 2009;
KRAUSE et al. 2010) and in vitro (WITTE et al. 2006) examined magnesium based
materials and pitting corrosion could be seen for LAE442 alloys in study XII as well,
with different occurrence depending on the pretreatment of the material and the
resulting grain size. The used nails were manufactured from extruded material which
provides a fine grain size which typically leads to a slower and more homogeneous
degradation of the material compared to as-casted material (ALVAREZ-LOPEZ et al.
2009). The slow degradation of the intramedullary nailing system caused acceptable
clinical impairment and radiological visible bone remodeling properties were adequate
as well. Bone remodeling processes in general were expected, as the implantation
process itself causes tissue reactions and the degradation process and the corrosion
products, respectively influence the surrounding bone as well. These influences were
49
supposed to be different for degradable materials compared with inert materials like
surgical steel or titanium. Newly formed bone was seen around the screws two to four,
which indicates a good osteoconductivity. However, a small gap could be observed
around screw 1, and it remained unclear if this gap was caused by gas cavities or
mechanical loosening of the screw. Inflammatory reactions were insignificant and only
visible in small areas of fibrous tissue predominantly around the central nails (studies
XXI and XXV). A low immunogenic potential was observed in former studies as well
(study XVI) and was stated by other authors in vitro (ROTH et al. 2014).
Beside the local biocompatibility, systemic toxicology might occur due to corrosion
products. Therefore, organ samples of excretory organs were examined histologically
and by means of inductively coupled plasma mass spectrometry for detection of
alloying elements. For rare earth elements, in the liver approximately ten times higher
values could be observed in the LAE442 group compared to the surgical steel group
(study XXV), in kidney and spleen, the values were approximately 2 and 3 fold
increased, respectively. In the kidney, even magnesium and aluminum were
significantly increased. However, it cannot be finally stated, if the increased element
concentrations are clinically relevant. Histologically, no pathological findings could be
observed in hematoxylin eosin stained organ samples. In conclusion, the magnesium
nails showed a very slow degradation rate combined with acceptable clinical,
radiographical and histological compatibility and might be an alternative to
conventional implant material from the perspective of biocompatibility although the
degradation rate was expected to be faster and in the present material the time until
complete degradation cannot be predicted precisely. Additionally, further examinations
concerning possible long term toxicity have to be performed.
50
A problem concerning the comparison of corrosion rates of different studies occurs
because the in vivo degradation rates can differ in dependence to the implant location
as well as between in vivo and in vitro studies (WITTE et al. 2006; BOBE et al. 2013;
SANCHEZ et al. 2014). In the examination of the MgCa0.8 and ZEK100 screws in
rabbit tibiae the screw heads of both examined materials degraded faster than the
parts which were located in the cortical bone (studies II and XVI). For the alloy LAE442
it could be stated that implants, which were manufactured from the same basic
material, showed different degradation rates and corresponding biocompatibility in
dependence to the implant location as well (study XIX). LAE442 plates which were
fixed on rabbit tibiae showed, in contrast to intramedullary implanted pins, a high
corrosion rate with huge amounts of gas formation and insufficient biocompatibility,
whereas the intramedullary implanted pins degraded very slowly combined with
adequate bone remodeling properties. Similar findings were observed for
intramedullary nailing systems in sheep as well, were gas cavities could be
predominantly observed around the heads of the interlocking screws, which were in
contact to the overlaying soft tissue. Location depending degradation rates were
observed in other studies as well. WILLBOLD et al. (2013) could show that Mg-based
implant materials (alloy RS66 which contained 6wt%Zn, 1wt%Y, and <1wt% Ce and
Zr) in vivo degraded faster in subcutaneous location compared to intramuscular or
cortical regions. The degradation rate obviously depends on the surrounding tissue
and the blood flow, and can differ in multiple parameters. In vitro studies showed that
especially the pH (NG et al. 2010; EVERTZ et al. 2013), different ions (WALKER et al.
2012; EVERTZ et al. 2013), the content of proteins (EVERTZ et al. 2013) as well as
possible mechanical stresses (DENKENA et al. 2013) can influence the degradation
rate of Mg-based implant materials. While an increasing pH decreases the corrosion,
higher amounts of chloride for example increase it. Another cause for different
51
degradation rates between plates and nails might be the application in different animal
models. The metabolic rate is slower in rabbits than in sheep and therewith degradation
rates might differ as well. The huge amount of different influencing parameters on
magnesium corrosion even causes the problem that a comparison between different
studies is almost impossible. First results of clinical studies with Magnetix®-screws in
humans did not show clinical problems (WINDHAGEN et al. 2013). The decreased
metabolic rate in humans might even decrease the degradation rate of magnesium
based implants and slow down gas formation correlated with clinical problems. Further
studies are necessary to improve and expand applications for promising magnesium
based implant materials.
5.4. Biomechanical implant requirements for fracture fixation in weight
bearing bones
VI. Reifenrath, J., Gottschalk, D., Angrisani, N., Besdo, S., Meyer-Lindenberg, A.:
Axial forces and bending moments in the loaded rabbit tibia in vivo; Acta Vet. Scand.,
2012, 54, 21, doi:10.1186/1751-0147-54-21
XX. Reifenrath, J., Angrisani, N., Lalk, M., Besdo, S.: Replacement, refinement and
reduction: necessity of standardization and computational models for long bone
fracture repair in animals, J Biomed. Mater. Res. A., 2014, 102, 8, p. 2884-900
XXI. Rössig, C., Angrisani, N., Besdo, S., Damm, N.B., Badenhop, M., Fedchenko,
N., Helmecke, P., Seitz, J.M., Meyer-Lindenberg, A., Reifenrath, J.: Magnesium-based
intramedullary nailing system in a sheep model: Biomechanic evaluation and first in
vivo results, J. Vet. Sci. Med. Diagn. 2014, 4, 1, doi:10.4172/2325-9590.1000150
52
In addition to biocompatibility mechanical parameters are key components for implant
materials which are developed for the use in weight bearing bones. For their
assessment the knowledge of the occurring forces and bending moments within the
scope of application as well as the mechanical characteristics of the material is
necessary to avoid implant failure or to calculate the interfragmentary movement. The
rabbit tibia is a common used model for the investigation of fracture healing and fixation
materials in weight bearing bones (WANG et al. 2005; SUMITOMO et al. 2008;
LECRONIER et al. 2012). However, in contrast to the sheep model, literature data
concerning mechanical parameters in this location were very rare. An indirect method
to calculate developing forces was described by GUSHUE et al. (2005). They used
infrared light emitting diodes which were attached to surgically fixed pins in the rabbit
hind leg combined with force measurement plates for their calculations. Direct
measurement methods were only described for humans (SEIDE et al. 2004; TAYLOR
et al. 2004) and sheep (MORA a. FORRIOL 2000; HELLER et al. 2005; GRASA et al.
2010). Therefor it was the aim of study VI to evaluate telemetrically the in vivo axial
forces, bending moments and bending angles in the rabbit tibia with weight bearing in
free physiological movement. It could be observed that axial forces decreased
continuously during the postoperative follow up due to callus formation in the
osteotomy gap. However, bending moments decreased only slightly. Fibrous tissue
and beginning osseous callus formation apparently better beared up against axial
forces than bending moments. Additionally, the results of this study showed that body
weight correlated axial forces in rabbit tibiae exceeded body weight correlated axial
forces in sheep tibiae and differed from indirectly correlated data by GUSHUE et al.
(2005). Axial loads and bending moments in rabbit tibiae were even more closely to
the human situation than axial loads and bending moments in the sheep tibia.
Nevertheless, a complete comparison remained difficult, as measured literature data
53
in sheep were incomplete concerning all occurring bending moments in the tibia and
the measured sheep walked on treadmills (CORDEY et al. 1980) in contrast to free
physiological movement in our study which allowed changes in speed and directions
during the measurement period. Similar approaches for the calculation of the
interfragmentary movement in a rat osteotomy model during a gait cycle free
movement were performed in another research group; WEHNER et al. (2010)
developed a numerical musculoskeletal model of the hind limb by the use of inverse
dynamics and calculated internal forces and moments in the rat femur. In this
calculation, forces in the femur reached 6 to 7 times bw in dependence to the location
which is much higher than the values we measured in the rabbit tibia (2 times bw) in
study VI. However, for studies in rat fracture models, predominantly the femur is used.
In contrast, in rabbit and sheep fracture models, the tibia is the most common used
bone (study XX). The multiple differences in used models and bones and the lack of a
consistent framework or generally accepted guidelines limit an effective comparison of
literature data.
The calculated load data from rabbit tibia and literature data for the sheep tibia
combined with additional µ-computed tomographical scans of both bone models
served as basis for finite element simulations. These simulations were used for the
comprehensive assessment, if different material and design properties of degradable
Mg-based osteosynthesis-systems might be mechanical sufficient for the required
application in the rabbit and sheep tibia osteotomy models. For the sheep tibia model,
first results showed that the less biomechanical stiffness and ultimate strength of
magnesium-based nails in comparison to surgical steel or titanium based nails might
reduce undesired stress shielding effects (VAN LOON et al. 1999; BE'ERY-
LIPPERMAN a. GEFEN 2006). Additional degradation over time (18% in 24 weeks)
can increase this positive effect. However, finite element simulation, which can detect
54
highest strain areas in implant and bone (LUTZ a. NACKENHORST 2012; TIOSSI et
al. 2013) showed that initial strength might be borderline for fracture fixation in weight
bearing bones (BESDO et al. 2013). A calculated sufficient stability of the assembly for
full load bearing was only assumed four weeks after fracture fixation due to an ongoing
callus formation with an increase of bending stiffness from 11% to 66% of the bone-
nail assembly in four-point bending test (study XXI). In the simulations, the increasing
load bearing of the healing fracture gap was superior to the decrease in stiffness
caused by degradation of the implant material. According to these first finite element
simulations, full load bearing cannot be recommended in fracture fixation with the
examined Mg-based intramedullary nailing systems during the first four weeks
postoperatively. At the current state of research, magnesium based intramedullary
nails as well as plate screw systems cannot be recommended for the use in clinical
studies. Especially in high loaded applications material properties have to be
considered as critical. Nevertheless applications with lower load (e.g. craniofacial
surgery) are a promising possible target area.
5.5. Comprehensive discussion of used animal models for the investigation of
biomaterials for orthopedic applications
In general, animal models are essential for the investigation of biomaterials prior to
clinical studies to avoid clinical failure of the material. However, no animal model can
precisely predict the material behavior in the target organism, which is predominantly
the human being or, in the field of veterinary medicine, companion animals like dogs
and cats. Even the special field of application can influence biocompatibility and
applicability of a biomaterial. Nevertheless, general properties and local reactions to
55
different materials can be examined in small and large animal models with following
extrapolation to the situation in other animals or humans.
It is necessary to clearly define the particular research question and to use an animal
model, which is reproducible and reflects the particular situation being evaluated
(DRESPE et al. 2005; PEARCE et al. 2007; CORRALES et al. 2008; MILLS a.
SIMPSON 2012). Additionally, prior to an animal experiment, the three R´s have to be
considered: to Reduce, Refine or Replace the animal experiments. In the examination
of implant materials, especially magnesium alloys, no in vitro model exists, which can
precisely predict implant degradation and biocompatibility. Therefore, material´s
examination in animal models is irreplaceable. In the present studies, predominantly
the rabbit model was used, although the mouse as small animal model (study XI) and
the sheep as large animal model (study XXI) were also implemented in some studies.
While some authors recommended to perform first in vivo studies of degradable
biomaterials in small animal models like mice and rats (ZHANG et al. 2009;
CASTELLANI et al. 2011; KRAUS et al. 2012; LINDTNER et al. 2013; WALKER et al.
2014b), other research groups prefer the sheep (WILLBOLD et al. 2013) or the rabbit
(WITTE et al. 2007b; LI et al. 2008; WITTE et al. 2010; WONG et al. 2010; ZHANG et
al. 2010). In our studies, different factors were considered in the choice of the animal
model, like availability, housing, ease of handling, costs, ethics, background data and
susceptibility to the disease (AN a. FRIEDMAN 1998). In biomaterial research,
especially inflammatory reactions to the material are of special interest as well as
degradation, when implanted in subcutaneous tissue. In the present studies, the
mouse model was only used for the investigation of osteoconductivity of magnesium.
In contrast to subcutaneous location, an intravascular approach was used, which was
established in the research consortium before. Magnesium, titanium and glyconate
implants were introduced into intravenous catheters the tail vein was punctured in the
56
cranial third after manually compression in the anesthetized mice and the implants
were pushed into the vein. Degradation rate and tissue reactions could be observed µ-
computed tomographically and histologically (MUELLER et al. 2012). Other authors
used rats for first investigations of magnesium degradation rates in subcutaneous
location (AGHION et al. 2012; WALKER et al. 2014b) or even in orthotopic location
(ZHANG et al. 2009; CASTELLANI et al. 2011; KRAUS et al. 2012; LINDTNER et al.
2013). Predominantly a transcortical application of simple pin geometries in the femur
was used (CASTELLANI et al. 2011; KRAUS et al. 2012; LINDTNER et al. 2013).
However, rats and mice are very small and more complex implant geometries are
difficult. Additionally, for in vivo µ-computed tomography a high resolution is necessary.
The resolution of the used in vivo µ-computer tomograph in the present studies is
limited to 41µm, which is borderline for the investigation of small implants especially in
an osseous environment, because the density of bone and magnesium implants is very
similar. Therefore, in most studies the rabbit was used as alternative. Advantages were
the ease in handling and lower costs compared to sheep models, and on the other
hand the possibility to use larger implants than in rats and mice. Beside simple pin
geometries of various magnesium alloys, which were tested within the intramedullary
cavity (studies I, V, VII, VIII, IX, X, XIII, XIV, XV, XXII) simple functional implants like
screws and osteosynthesis plates could be examined as well (studies II, XVI, XVIII).
Pull out test of implanted screws with material test systems could be used to evaluate
the holding power of magnesium based screws after different implantation times.
Smaller geometries, which would have been necessary for mice and rats, limit these
evaluations techniques.
Especially the rabbit tibia model could be established in the last years in our research
group and a comparison between different materials was possible due to a
standardized approach and implantation in the medullary cavity. The measurement of
57
force and bending moment data in physiological movement (study VI) secondary
offered the possibility for the establishment of simulation models. These models can
be used for the adjustment of designs in osteosynthesis systems prior to the evaluation
in the animal model. Mechanical parameters can be calculated with these simulations,
which may contribute to less material failure in the in vivo situation. More detailed
information about animal models in fracture repair is given in study XXI. In magnesium
research, many other authors used the rabbit as animal model as well (WITTE et al.
2007b; LI et al. 2008; WITTE et al. 2010; WONG et al. 2010; ZHANG et al. 2010).
However, for more complex implant systems like interlocked intramedullary nailing
systems, even the rabbit model is too small. For this purpose the sheep model, which
is the predominately used large animal model in fracture repair (studies XXI, XXIV,
XXV), was used. Many different fixation techniques like osteosynthesis plates or
intramedullary nails were examined in this model as well as bone healing in critical size
defects (TEIXEIRA et al. 2007; LU et al. 2009; MUELLER et al. 2009; KLEIN et al.
2010; NIEMEYER et al. 2010; PLECKO et al. 2012; TRALMAN et al. 2012). However,
the sheep model has some limitations; for in vivo µ-computed tomography, the sheep
was too large and the resolution in clinically used computed tomography was very
imprecise for the evaluation of implant degradation in vivo (study XXI). Detailed
information about implant volume loss and bone remodeling properties could only be
achieved after euthanasia by the use of additional methods like ex vivo µ-CT and
histology. Also variances among the individuals were greater than in the used small
animals (mice and rabbits). For more reliable statistical results the number of used
animals should be increased, but costs and efforts of care often limit this option.
The choice of the animal model and the implant location influence the implant material.
Substantial differences in implant degradation were observed for the same implant
58
material LAE442 in dependence to the implant location (plate-screw-system degraded
much faster than the intramedullary nailing system). It cannot be clearly stated, if these
differences only were caused by the variances in the surrounding tissue or additionally
by variances in the used animal model. Whereas the plate-screw systems were
implanted in the rabbit model, the intramedullary nailing system was tested in the
sheep model. Differences in the metabolic rate, which is faster in smaller animals, are
a possible explanation for decreased gas formation as well. A comparative study of the
degradation of magnesium based implant materials between different animal models
does not exist yet.
In conclusion, the use of the animal model has to be carefully chosen prior to the study
and should first of all implement the precise research question and second the
available possibilities in handling, housing as well as methods for the investigation.
Especially for the development of biomaterials for fracture fixation devices at least for
first examination of biocompatibility and calculation of suitable mechanical stabilities
the rabbit is a very convenient animal model, whereas for further studies of more
complex implant systems the sheep model is more suitable.
Beside these animal models, mice or rats should always be taken into account,
especially for first preliminary studies of new biodegradable materials.
59
6. Summary
In vivo evaluation of degradable magnesium alloys as orthopedic implant
material in suitable animal models
Janin Reifenrath
Until today, commonly used implant materials in fracture repair are permanent and
need to be removed after the healing process. In the present studies, biodegradable
magnesium based implant materials were tested in different animal models for the use
as orthopedic implant material in weight bearing applications. Therefore, mechanical
strength is required beside a good biocompatibility during the degradation process. As
in vitro studies are still not able to represent the complex in vivo situation, examinations
in animal models are indispensable prior to clinical studies. In this interdisciplinary
collaborative research on the development of magnesium-based implant materials for
loaded applications, material and engineering scientists, bio-mechanists, and
orthopedic surgeons were implemented. First promising materials were carved out by
the use of clinical, radiographical, (µ-) computed tomographical and histological
methods as well as biomechanical approaches, predominantly in the rabbit model.
These materials, especially LAE442, were afterwards evaluated in application-oriented
studies as osteosynthesis system (plate-screw system and interlocked intramedullary
nailing system). While the intramedullary nailing system showed an adequate
biocompatibility and very slow implant degradation, the plate screw-system caused
massive clinical problems with gas formation and lameness and could not be
recommended for the clinical use in the current state. However, the reason for the
differences in implant degradation and resultant tissue reactions in the implant
surrounding could not be finally resolved. Magnesium degradation is influenced by
various factors, including pretreatments which can influence the surface structure,
60
mechanical stresses during the implantation and differences in the surrounding tissue,
especially ion concentrations. Although the intramedullary nailing systems were very
promising, additional finite element simulations showed that the mechanical strength
is borderline for fracture fixation without an additional stabilization within the first
weeks. Further studies should clarify whether positive effects (like reduced stress
shielding over time) are superior to the restriction in application.
The evaluation in animal models still remains indispensable, as the degradation in vitro
differs from the degradation in vivo. Until now, no system can predict the in vivo
situation. Especially the rabbit is a very commonly used and suitable animal model. It
combines the ease in housing and handling requirements with the possibility of
application oriented testing of implant materials by the use of various imaging
techniques, including µ-computed tomography as well as simple biomechanical test.
Only more complex interlocked intramedullary nailing systems were tested in the
sheep as large animal model. However, differences in the metabolic rate as well as
differences in the bone structure always have to be taken into account, when results
are extrapolated to other animals or the human medicine.
Considering the studies together, low inflammatory reactions of the tissues
surrounding magnesium based implants are beneficial. If the degradation process is
slow, gas which occurs during the degradation process can be resorbed without clinical
problems. The use as an osteosynthesis system especially in high loaded applications
has to be considered as problematic due to a borderline mechanical stability and high
amounts of material. However, smaller implants or lower loaded areas are very
promising applications for magnesium based implant materials.
61
7. References
AGHION, G. LEVY u. S. OVADIA (2012): In vivo behavior of biodegradable Mg–Nd–Y–Zr–Ca alloy. J Mater Sci: Mater Med 23, 805-812 ALBREKTSSON; T. u. C. JOHANSSON (2001): Osteoinduction, osteoconduction and osseointegration. Eur Spine J 10 Suppl 2, S96-101 ALVAREZ-LOPEZ, M., D. PEREDA, J. A. DEL VALLE, M. FERNANDEZ-LORENZO, M. C. GARCIA-ALONSO, O. A. RUANO u. M. L. ESCUDERO (2009): Corrosion behaviour of AZ31 magnesium alloy with different grain sizes in simulated biological fluids. Acta Biomater 6, 1763-1771 AN, u. R. J. FRIEDMAN (1998): Animal models in orthopaedic research. CRC Press, Boca Raton, ISBN 0-8493-2115-8 AN, Y.H. u. MARTIN, K. L. (2003): Handbook of Histology Methods for Bone and Cartilage, Humana Press; Totowa NJ, ISBN 1617372773 AVEDESIAN, M.M. u. BAKER, H. (1999): ASM specialty handbook, ASM International, Materials Park, Ohio,ISBN 0-87170-657-1 BABIKER, H. (2013): Bone graft materials in fixation of orthopaedic implants in sheep. Dan Med J 60, B4680 BE'ERY-LIPPERMAN, M. u. A. GEFEN (2006): A method of quantification of stress shielding in the proximal femur using hierarchical computational modeling. Computer Methods in Biomechanics and Biomedical Engineering 9, 35-44 BERRY, M. (2008): Bioresorbable composite materials for orthopaedic devices. Med Device Technol 19, 69-70, 72 BESDO, S., A.-K. KRÜGER, C. RÖSSIG, P. HELMECKE, H. WAIZY, P. WRIGGERS, N. ANGRISANI u. J. REIFENRATH (2013): Finite Element Study of Degradable Intramedullary Nails out of Magnesium Alloy for Ovine Tibiae. Biomedical Engineering / Biomedizinische Technik, DOI 10.1515/bmt-2013-4073 BHALLA, P., N. SINGLA u. D. K. DHAWAN (2010): Potential of lithium to reduce aluminium-induced cytotoxic effects in rat brain. Biometals 23, 197-206
62
BOBE, K., E. WILLBOLD, I. MORGENTHAL, O. ANDERSEN, T. STUDNITZKY, J. NELLESEN, W. TILLMANN, C. VOGT, K. VANO u. F. WITTE (2013): In vitro and in vivo evaluation of biodegradable, open-porous scaffolds made of sintered magnesium W4 short fibres. Acta Biomater 9, 8611-8623 BÖSTMAN, O. u. H. PIHLAJAMÄKI (2000): Clinical biocompatibility of biodegradable orthopaedic implants for internal fixation: a review. Biomaterials 21, 2615-2621 BUHA, J. (2008): Natural ageing in magnesium alloys and alloying with Ti. J Mater Sci 43, 1220-1227 CASTELLANI, C., R. A. LINDTNER, P. HAUSBRANDT, E. TSCHEGG, S. E. STANZL-TSCHEGG, G. ZANONI, S. BECK u. A.-M. WEINBERG (2011): Bone-implant interface strength and osseointegration: Biodegradable magnesium alloy versus standard titanium control. Acta Biomater 7, 432-440 CHAO, E. Y., H. T. ARO, D. G. LEWALLEN u. P. J. KELLY (1989): The effect of rigidity on fracture healing in external fixation. Clin. Orthop. Relat. Res., 24-35 CHAYA, A. (2014) Degradable magnesium plates and screws for bone fracture fixation A 6th Symposium on biodegradable metals, Maratea, Italy, inviv 5 CHEN, C.H., V. B.-H. SHYU, Y.-C. CHEN, H.-T. LIAO, C.-J. LIAO u. C.-T. CHEN (2013): Reinforced bioresorbable implants for craniomaxillofacial osteosynthesis in pigs. Br J Oral Maxillofac Surg 51, 948-952 CIPITRIA, A., J. C. REICHERT, D. R. EPARI, S. SAIFZADEH, A. BERNER, H. SCHELL, M. MEHTA, M. A. SCHUETZ, G. N. DUDA u. D. W. HUTMACHER (2013): Polycaprolactone scaffold and reduced rhBMP-7 dose for the regeneration of critical-sized defects in sheep tibiae. Biomaterials 34, 9960-9968 CLAES, L. E. u. C. A. HEIGELE (1999): Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing. J Biomech 32, 255-266 CORDEY, J., M. SCHNETZER, J. BRENNWALD, P. REGAZZONI u. S. M. PERREN (1980): Direct in vivo Measurements of Torque and Bending in Sheep Tibiae in: UHTHOFF, H. K. u. E. STAHL (Hrsg.): Current concepts of internal fixation of fractures, Springer Verlag, Berlin, New York
63
CORRALES, L. A. S. MORSHED, M. BHANDARI u. T. MICLAU (2008): Variability in the assessment of fracture-healing in orthopaedic trauma studies. J Bone Joint Surg Am 90, 1862-1868 DÉJARDIN, L. M., L. P. GUIOT u. D. J. F. VON PFEIL (2012): Interlocking nails and minimally invasive osteosynthesis. Vet. Clin. North Am. Small Anim. Pract. 42, 935-62 DENKENA, B., J. KÖHLER, J. STIEGHORST, A. TURGER, J. M. SEITZ, D. R. FAU, L. WOLTERS, N. ANGRISANI, J. REIFENRATH, P. HELMECKE (2013): Influence of stress on the degradation behavior of Mg LAE442 implant systems. Elsevier B.V. 5, 189-195 DOI 10.1016/j.procir.2013.01.038 DEPPRICH, R., M. OMMERBORN, H. ZIPPRICH, C. NAUJOKS, J. HANDSCHEL, H.-P. WIESMANN, N. R. KÜBLER u. U. MEYER (2008): Behavior of osteoblastic cells cultured on titanium and structured zirconia surfaces. Head Face Med 4, 29 DISEGI J. A. u. L. ESCHBACH (2000): Stainless steel in bone surgery. Injury 31, 2-6 DONATH, K. (1995): Preparation of histologic sections, Exakt Kulzer Publication http://www.exakt.de/fileadmin/user_upload/PDF/Preparation_of_Histologic_Sections.pdf DRESPE I. H., G. K. POLZHOFER, A. S. TURNER u. J. N. GRAUER (2005): Animal models for spinal fusion. Spine J 5, 209-216 ENGELBERG, I. u. J. KOHN (1991): Physico-mechanical properties of degradable polymers used in medical applications: a comparative study. Biomaterials 12, 292-304 EVERTZ, F., H. HAUSER, P. P. MÜLLER, M. KIETZMANN, H. J. MAIER u. B. GLASMACHER (2013): Magnesium as a biomaterial and its biological interactions. Biomed Tech, Berlin, DOI 10.1515/bmt-2013-4066 GHOSH, S., A. SHARMA u. G. TALUKDER (1992): Zirconium. An Abnormal Trace Element in Biology. Biological trace element research 35, 247-271 GOGOLEWSKI, S. (2000): Bioresorbable polymers in trauma and bone surgery. Injury 31, 28-32 GRADINGER, R. u. H. GOLLWITZER (2006): Ossäre Integration.
64
Springer Medizin, Berlin, Heidelberg GRANDJEAN, E. M. u. J.-M. AUBRY (2009a): Lithium: updated human knowledge using an evidence-based approach: Part I: Clinical efficacy in bipolar disorder. CNS Drugs 23, 225-240 GRANDJEAN, E. M u. J.-M. AUBRY (2009b): Lithium: updated human knowledge using an evidence-based approach: part III: clinical safety. CNS Drugs 23, 397-418 GRASA, J., M. J. GÓMEZ-BENITO, L. A. GONZÁLEZ-TORRES, D. ASIAÍN, F. QUERO u. J. M. GARCÍA-AZNAR (2010): Monitoring in vivo load transmission through an external fixator. Ann Biomed Eng 38, 605-612 GUNDBERG, C. M. (2003): Matrix proteins. Osteoporos Int 14 Suppl 5, S37-40; discussion S40-2 GUSHUE, D. L., J. HOUCK u. A. L. LERNER (2005): Rabbit knee joint biomechanics: motion analysis and modeling of forces during hopping. J Orthop Res 23, 735-742 GU, X., Y. ZHENG, Y. CHENG, S. ZHONG u. T. XI (2009): In vitro corrosion and biocompatibility of binary magnesium alloys. Biomaterials 30, 484-498 GU, X. N. u. Y.-F. ZHENG (2010): A review on magnesium alloys as biodegradable materials. Front. Mater. Sci. China 4, 111-115 GU, X. N., W. R. ZHOU, Y. F. ZHENG, Y. CHENG, S. C. WEI, S. P. ZHONG, T. F. XI u. L. J. CHEN (2010): Corrosion fatigue behaviors of two biomedical Mg alloys - AZ91D and WE43 - In simulated body fluid. Acta Biomater 6, 4605-4613 HABIBOVIC, P. u. K. DE GROOT (2007): Osteoinductive biomaterials--properties and relevance in bone repair. J Tissue Eng Regen Med 1, 25-32 HÄNZI, A. C., P. GUNDE, M. SCHINHAMMER u. P. J. UGGOWITZER (2009): On the biodegradation performance of an Mg-Y-RE alloy with various surface conditions in simulated body fluid. Acta Biomater 5, 162-171 HELLER, M. O., G. N. DUDA, R. M. EHRIG, H. SCHELL, P. SEEBECK u. TAYLOR R. W. (2005):
65
Muskuloskeletale Belastungen im Schafshinterlauf: Mechanische Rahmenbedingungen der Heilung. Mat.-wiss. u. Werkstofftech. 36, 775-780 HE, Y., H. ZHANG u. J. CUI (2010): Effects of Pre-Ageing Treatment on Subsequent Artifcial Ageing Characteristics of an Al-1.01Mg-0.68Si-1.78Cu Alloy. J. Mater. Sci. Technol. 26 HIRANO, S. u. K. T. SUZUKI (1996): Exposure, metabolism, and toxicity of rare earths and related compounds. Environ. Health Perspect. 104 Suppl 1, 85-95 HISTING, T., P. GARCIA, J. H. HOLSTEIN, M. KLEIN, R. MATTHYS, R. NUETZI, R. STECK, M. W. LASCHKE, T. WEHNER, R. BINDL, S. RECKNAGEL, E. K. STUERMER, B. VOLLMAR, B. WILDEMANN, J. LIENAU, B. WILLIE, A. PETERS, A. IGNATIUS, T. POHLEMANN, L. CLAES u. M. D. MENGER (2011): Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting. Bone 49, 591-599 HOFMANN, G. O. (1995): Biodegradable implants in traumatology: a review on the state-of-the-art. Arch Orthop Trauma Surg 114, 123-132 HORT, N., Y. HUANG, D. FECHNER, M. STÖRMER, C. BLAWERT, F. WITTE, C. VOGT, H. DRÜCKER, R. WILLUMEIT, K. U. KAINER u. F. FEYERABEND (2009): Magnesium alloys as implant materials - Principles of property design for Mg-RE alloys. Acta Biomater 6, 1714-1725 HUEHNERSCHULTE, T. A., A., KRAUSE, A., KRAUSE, CH., VON DER HOEH N., WINDHAGEN, H. u. MEYER-LINDENBERG, A. (2009): Comparison of the biomechanical properties of the Magnesium alloys ZEK100, AX30, LAE442 and MgCa0.8 after 3 and 6 month implantation in rabbit tibiae. HUEHNERSCHULTE, T. A. N. ANGRISANI, D. RITTERSHAUS, D. BORMANN, H. WINDHAGEN u. A. MEYER-LINDENBERG (2011): In vivo corrosion of two novel magnesium alloys ZEK100 and AX30 and their mechanical suitability as biodegradable implants. Materials 4, 1144-1167 IGNATIUS, A. A. u. L. E. CLAES (1996): In vitro biocompatibility of bioresorbable polymers: poly(L, DL-lactide) and poly(L-lactide-co-glycolide). Biomaterials 17, 831-839 KAESE, V. (2002): Beitrag zum korrosionsschützenden Legieren von Magnesiumwerkstoffen. VDI, Düsseldorf
66
KANNAN, M. B. u. R. K. S. RAMAN (2008): In vitro degradation and mechanical integrity of calcium-containing magnesium alloys in modified-simulated body fluid. Biomaterials 29, 2306-2314 KLEIN, C., C. SPRECHER, B. A. RAHN, J. GREEN u. C. A. MÜLLER (2010): Unreamed or RIA reamed nailing: an experimental sheep study using comparative histological assessment of affected bone tissue in an acute fracture model. Injury 41 Suppl 2, 32-7 KOMATSU, S. Y., M. IKEDA, U. MORI u. M. ABE (2004): Increase in resistivity of Mg-Zn alloys by low temperature aging. Keikinzoku 54, 131-136 KRAUSE, A. (2008): Untersuchung der Degradation und Biokompatibilität von degradablen, intramedullären Implantaten auf Magnesiumbasis im Kaninchenmodell. Hannover, Stiftung Tierärztliche Hochschule Hannover, Dissertation. KRAUSE, A., C. HACKENBROICH, N. VON DER HÖH, S. WAGNER, D. BORMANN, T. HASSEL, H. WINDHAGEN u. A. MEYER-LINDENBERG (2005): Rare earth containing magnesium alloys as degradable intramedullar implants in rabbit tibiae. Biomaterialien 6, 190 KRAUSE, A., N. VON DER HÖH, D. BORMANN, C. KRAUSE, F.-W. BACH, H. WINDHAGEN u. A. MEYER-LINDENBERG (2010): Degradation behaviour and mechanical properties of magnesium implants in rabbit tibiae. J Mater Sci 45, 624-632 KRAUS, T., S. F. FISCHERAUER, A. C. HÄNZI, P. J. UGGOWITZER, J. F. LÖFFLER u. A. M. WEINBERG (2012): Magnesium alloys for temporary implants in osteosynthesis: In vivo studies of their degradation and interaction with bone. Acta Biomater 8, 1230-1238 KUWAHARA, H., Y. AL-ABDULLAT, M. OHTA, K. IKEUCHI, N. MAZAKI u. T. AIZAWA (2000): Surface reaction of magnesium in Hank's solutions. Mater Sci. Forum 350-351, 349-358 LÅFTMAN, P., O. S. NILSSON, O. BROSJÖ u. L. STRÖMBERG (1989): Stress shielding by rigid fixation studied in osteotomized rabbit tibiae. Acta Orthop Scand 60, 718-722 LAMBOTTE, A. (1932): L’utilisation du magnesium comme materiel perdu dans l’osteosynthèse. Bull Mém Soc Nat Chir 28, 1325-1334
67
LANDES, C., A. BALLON, S. GHANAATI, D. EBEL, D. ULRICH, U. SPOHN, U. HEUNEMANN, R. SADER u. R. JAEGER (2013): Evaluation of the Fatigue Performance and Degradability of Resorbable PLDLLA-TMC Osteofixations. Open Biomed Eng J 7, 133-146 LECRONIER, D. J., J. S. PAPAKONSTANTINOU, V. GHEEVARUGHESE, C. D. BERAN, N. E. WALTER u. P. J. ATKINSON (2012): Development of an interlocked nail for segmental defects in the rabbit tibia. Proc Inst Mech Eng H 226, 330-336 LEE, D. B. M., M. ROBERTS, C. G. BLUCHEL u. R. A. ODELL (2010): Zirconium: biomedical and nephrological applications. ASAIO J. 56, 550-556 LI, Z., X. GU, S. LOU u. Y. ZHENG (2008): The development of binary Mg-Ca alloys for use as biodegradable materials within bone. Biomaterials 29, 1329-1344 LI, Y., Z. M. ZHANG u. Y. XUE (2011): Influence of aging on microstructure and mechanical properties of AZ80 and ZK60 magnesium alloys. Trans. Nonferrous Met. Soc. China 21, 739-744 LINDTNER, R. A., C. CASTELLANI, S. TANGL, G. ZANONI, P. HAUSBRANDT, E. K. TSCHEGG, S. E. STANZL-TSCHEGG u. A.-M. WEINBERG (2013): Comparative biomechanical and radiological characterization of osseointegration of a biodegradable magnesium alloy pin and a copolymeric control for osteosynthesis. J Mech Behav Biomed Mater 28, 232-243 LIU, C., Y. XIN, G. TANG u. P. K. CHU (2007): Influence of heat treatment on degradation behavior of bio-degradable die-cast AZ63 magnesium alloy in simulated body fluid. Mater Sci Eng A 456, 350-357 LU, Y., B. NEMKE, D. M. LORANG, R. TRIP, H. KOBAYASHI u. M. D. MARKEL (2009): Comparison of a new braid fixation system to an interlocking intramedullary nail for tibial osteotomy repair in an ovine model. Vet Surg 38, 467-476 LUTZ, W. u. U. NACKENHORST (2012): Numerical investigations on the osseointegration of uncemented endoprostheses based on bio-active interface theory. Computational Mechanics 50, 367-381 MAJOLA, A., S. VAINIONPÄÄ, K. VIHTONEN, J. VASENIUS, P. TÖRMÄLÄ u. P. ROKKANEN (1992): Intramedullary fixation of cortical bone osteotomies with self-reinforced polylactic rods in rabbits.
68
International orthopaedics 16, 101-108 MARTÍNEZ, M., C. BOZZINI, M. I. OLIVERA, G. DMYTRENKO u. M. I. CONTI (2011): Aluminum bone toxicity in immature rats exposed to simulated high altitude. J. Bone Miner. Metab. 29, 526-534 MARTINI, L.,M. FINI, G. GIAVARESI u. R. GIARDINO (2001): Sheep model in orthopedic research: a literature review. Comp. Med. 51, 292-299 MARTINIAKOVÁ, M., B. GROSSKOPF, R. OMELKA, M. VONDRÁKOVÁ u. M. BAUEROVÁ (2006): Differences among species in compact bone tissue microstructure of mammalian skeleton: use of a discriminant function analysis for species identification. J. Forensic Sci. 51, 1235-1239 MCBRIDE, E. D. (1938): Absorbable metal in bone surgery. J Am Med Assoc 111, 2464-2467 MCKEE, M. D., C. E. PEDRAZA u. M. T. KAARTINEN (2011): Osteopontin and wound healing in bone. Cells Tissues Organs 194, 313-319 MILLER, D. L. u. T. GOSWAMI (2007): A review of locking compression plate biomechanics and their advantages as internal fixators in fracture healing. Clin. Biomech. 22, 1049-1062 MILLS, L. A. u. A. H. R. W. SIMPSON (2012): In vivo models of bone repair. J Bone Joint Surg Br 94, 865-874 MORA, G. u. F. FORRIOL (2000): Mechanical analysis of the healing of different osteotomies fixed externally. Int Orthop 24, 295-298 MUELLER, W. D. (2007): Magnesium and its alloys as degradable biomaterials. Corrosion studies using potentiodynamic and EIS electrochemical techniques. Mater Res 10, 5-10 MUELLER, W. D., S. ARNOLD, M. BADAR, D. BORMANN, F.-W. BACH, A. DRYNDA, A. MEYER-LINDENBERG, H. HAUSER u. M. PEUSTER (2012): Histological and molecular evaluation of iron as degradable medical implant material in a murine animal model. J Biomed. Mater. Res. A 100, 2881-9 MUELLER, C. A., V. SCHLEGEL, F. HOEGEL, C. ECKHARDT, U. SCHLEGEL, B. A. RAHN, U. PFISTER u. N. P. SUEDKAMP (2009):
69
Cortical perfusion and local fat occlusion after intramedullary nailing of the ovine tibia-comparison of different surgical procedures. Injury 40, 760-766 NAGELS, J., M. STOKDIJK u. P. M. ROZING (2003): Stress shielding and bone resorption in shoulder arthroplasty. J Shoulder Elbow Surg 12, 35-39 NAKATSUGAVA, I., S. KAMADO, Y. KOJIMA, R. NINOMIYA u. K. KUBOTA (1998): Corrosion of magnesium alloys containing rare earth elements. Corrosion Reviews 16, 139-158 NG, W.F., K. Y. CHIU u. F. T. CHENG (2010): Effect of pH on the in vitro corrosion rate of magnesium degradable implant material. Mater. Sci. Eng. C., 898-903 NIEMEYER, P., T. S. SCHÖNBERGER, J. HAHN, P. KASTEN, J. FELLENBERG, N. SUEDKAMP, A. T. MEHLHORN, S. MILZ u. S. PEARCE (2010): Xenogenic transplantation of human mesenchymal stem cells in a critical size defect of the sheep tibia for bone regeneration. Tissue Eng. Part A 16, 33-43 OSTROWSKI, N., B. LEE, N. ENICK, B. CARLSON, S. KUNJUKUNJU, A. ROY u. P. N. KUMTA (2013): Corrosion protection and improved cytocompatibility of biodegradable polymeric layer-by-layer coatings on AZ31 magnesium alloys. Acta Biomater 9, 8704-8713 PARDO, A., M. C. MERINO, A. E. COY, R. ARRABAL, F. VIEJO u. E. MATYKINA (2008): Corrosion behaviour of magnesium/aluminium alloys in 3.5 wt.% NaCl. Corros Sci 50, 823-834 PARFITT , A. M. (1994): Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone. J Cell Biochem 55, 273-286 PEARCE, A. I., R. G. RICHARDS, S. MILZ, E. SCHNEIDER u. S. G. PEARCE (2007): Animal models for implant biomaterial research in bone: a review. Eur Cell Mater 13, 1-10 PIENKOWSKI, D., G. C. STEPHENS, T. M. DOERS u. D. M. HAMILTON (1998): Multicycle mechanical performance of titanium and stainless steel transpedicular spine implants. Spine 23, 782-788 PLECKO, M., N. LAGERPUSCH, B. PEGEL, D. ANDERMATT, R. FRIGG, R. KOCH, M. SIDLER, P. KRONEN, K. KLEIN, K. NUSS, P. GEDET, A. BÜRKI, S. J. FERGUSON, U. STOECKLE, J. A. AUER u. B. VON RECHENBERG (2012):
70
The influence of different osteosynthesis configurations with locking compression plates (LCP) on stability and fracture healing after an oblique 45° angle osteotomy. Injury 43, 1041-1051 POHLER, O. E. M. (2000): Unalloyed titanium for implants in bone surgery. Injury 31, 7-13 POLMEAR I. J. (1999): Grades and Alloys in: AVEDESIAN, M. M. u. H. BAKER (Hrsg.): Magnesium and magnesium alloys, ASM International, Materials Park, Ohio, 12-23 RAHIM, I. R., P. P. MUELLER, M. ROHDE u. H. HAUSER (2013): Evaluation and comparison of in vitro and in vivo degradation kinetics of magnesium. Biomed Engineer / Biomed Tech, DeGruyter, Berlin, doi: 10.1515/bmt-2013-4107 REIFENRATH, J., N. ANGRISANI, M. LALK u. S. BESDO (2014): Replacement, refinement, and reduction: Necessity of standardization and computational models for long bone fracture repair in animals. J Biomed Mater Res A 102, 2884-2900 REMENNIK, S., I. BARTSCH, E. WILLBOLD, F. WITTE u. D. SHECHTMAN (2011): New, fast corroding high ductility Mg–Bi–Ca and Mg–Bi–Si alloys, with no clinically observable gas formation in bone implants. Mat Sci Eng B-Solid 176, 1653-1659 REVELL, P. A., E. DAMIEN, X. ZHANG, P. EVANS u. C. HOWLETT (2004): The effect of magnesium ions on bone bonding to hydroxyapatite. Key Eng Mater 254-256, 447-450 ROKKANEN, P. U., O. BÖSTMAN, E. HIRVENSALO, E. A. MÄKELÄ, E. K. PARTIO, H. PÄTIÄLÄ, S. I. VAINIONPÄÄ, K. VIHTONEN u. P. TÖRMÄLÄ (2000): Bioabsorbable fixation in orthopaedic surgery and traumatology. Biomaterials 21, 2607-2613 ROTH, I., S. SCHUMACHER, T. BASLER, K. BAUMERT, J. M. SEITZ, F. EVERTZ,
P. P. MÜLLER, W. BÄUMER u. M. KIETZMANN (2014):
Magnesium corrosion particles do not interfere with the immune function of primary
human and murine macrophages.
Prog Biomater 12 doi:: 10.1007/s40204-014-0032-9
RUSSELL W. M. S. u. R. L. BURCH (1992): The principles of humane experimental technique. 2nd edition, UFAW, Hearts, UK SALDAÑA, L., A. MÉNDEZ-VILAS, L. JIANG, M. MULTIGNER, J. L. GONZÁLEZ-CARRASCO, M. T. PÉREZ-PRADO, M. L. GONZÁLEZ-MARTÍN, L. MUNUERA u. N. VILABOA (2007): In vitro biocompatibility of an ultrafine grained zirconium.
71
Biomaterials 28, 4343-4354 SALEH, K. J., I. THONGTRANGAN u. E. M. SCHWARZ (2004): Osteolysis: medical and surgical approaches. Clin. Orthop. Relat. Res.427, 138-147 SANCHEZ, A. H. M., B. J. C. LUTHRINGER, F. FEYERABEND u. R. WILLUMEIT (2014): Mg and Mg alloys: How comparable are in vitro and in vivo corrosion rates? - A Review. Acta Biomater 13, 16-31 SCHATZKER J. u. J. E. F. HOULTON (2002): Concepts of fracture stabilization in: SUMNER-SMITH, G. u. G. E. FACKELMAN (Hrsg.): Bone in Clinical Orthopedics, Thieme, Stuttgart, 327-347 SEIDE, K., N. WEINRICH, M. E. WENZL, D. WOLTER u. C. JÜRGENS (2004): Three-dimensional load measurements in an external fixator. J Biomech 37, 1361-1369 SONG, G. L. u. A. ATRENS (1999): Corrosion mechanisms of magnesium alloys. Adv Eng Mater 1, 11-33 SONG, G. L. u. A. ATRENS (2003): Understanding Magnesium Corrosion - A Framework for Improved Alloy Performance. Adv Eng Mater 5, 837-858 STAIGER,M., A. M. PIETAK, J. HUADMAI u. G. DIAS (2006): Magnesium and its alloys as orthopedic biomaterials: a review. Biomaterials 27, 1728-1734 STIFFLER, K. S. (2004): Internal fracture fixation. Clin Tech Small Anim Pract 19, 105-113 SUMITOMO, N., K. NORITAKE, T. HATTORI, K. MORIKAWA, S. NIWA, K. SATO u. M. NIINOMI (2008): Experiment study on fracture fixation with low rigidity titanium alloy: plate fixation of tibia fracture model in rabbit. J Mater Sci Mater Med 19, 1581-1586 TANAKA, T.u. T. KISHIMOTO (2014): The biology and medical implications of interleukin-6. Cancer Immunol Res 2, 288-294 TAYLOR, W. R., M. O. HELLER, G. BERGMANN u. G. N. DUDA (2004): Tibio-femoral loading during human gait and stair climbing.
72
J. Orthop. Res. 22, 625-632 TEIXEIRA, C. R., S. C. RAHAL, R. S. VOLPI, R. TAGA, T. M. CESTARI, J. M. GRANJEIRO, L. C. VULCANO u. M. A. CORREA (2007): Tibial segmental bone defect treated with bone plate and cage filled with either xenogeneic composite or autologous cortical bone graft. An experimental study in sheep. Vet Comp Orthop Traumatol 20, 269-276 THOMANN, M., C. KRAUSE, N. ANGRISANI, D. BORMANN, T. HASSEL, H. WINDHAGEN u. A. MEYER-LINDENBERG (2010a): Influence of a magnesium-fluoride coating of magnesium-based implants (MgCa0.8) on degradation in a rabbit model. J Biomed Mater Res A 93, 1609-1619 THOMANN, M., C. KRAUSE, D. BORMANN, N. VON DER HÖH, H. WINDHAGEN u. A. MEYER-LINDENBERG (2009): Comparison of the resorbable magnesium alloys LAE442 und MgCa0.8 concerning their mechanical properties, their progress of degradation and the bone-implant-contact after 12 months implantation duration in a rabbit model. Mat Sci Eng Tech 40, 82-87 THOMANN, M., VON DER HOEH N., D. BORMANN, D. RITTERSHAUS, C. KRAUSE, H. WINDHAGEN u. A. MEYER-LINDENBERG (2010b): Comparison of the cross sectional area, the loss in volume and the mechanical properties of LAE442 and MgCa0.8 as resorbable magnesium alloy implants after 12 months implantation duration. Mater Sci Forum 638-642, 675-680 TIOSSI, R., M. VASCO, L. LIN, H. CONRAD, O. BEZZON, R. RIBEIRO u. A. FOK (2013): Validation of finite element models for strain analysis of implant-supported prostheses using digital image correlation. Dent Mater 29, 788-796 TOPF, J. M. u. P. T. MURRAY (2003): Hypomagnesemia and hypermagnesemia. Rev Endocr Metab Disord 4, 195-206 TRALMAN, G., V. ANDRIANOV, A. AREND, P. MÄNNIK, R. T. KIBUR, K. NÕUPUU, D. UKSOV u. M. AUNAPUU (2012): A Novel Combined Method of Osteosynthesis in Treatment of Tibial Fractures: A Comparative Study on Sheep with Application of Rod-Through-Plate Fixator and Bone Plating. Anat Histol Embryol 42, 80-89 TSCHEGG, E. K., R. A. LINDTNER, V. DOBLHOFF-DIER, S. E. STANZL-TSCHEGG, G. HOLZLECHNER, C. CATELLANI, T. IMWINKELRIED u. A. WEINBERG (2011): Characterization methods of bone-implant-interfaces of bioresorbable and titanium implants by fracture mechanical means.
73
J Mech Behav Biomed Mater 4, 766-775 UHTHOFF H. K. u. M. FINNEGAN (1983): The effects of metal plates on post-traumatic remodelling and bone mass. J Bone Joint Surg Br 65, 66-71 VAN LOON, C. J., M. C. DE WAAL MALEFIJT, P. BUMA, N. VERDONSCHOT u. R. P. VETH (1999): Femoral bone loss in total knee arthroplasty. A review. Acta Orthop Belg 65, 154-163 VERBRUGGE J. (1934): Le matériel métallique résorbable en chirurgie osseuse. La Press Med 23, 460-465 WALKER, J., S. SHADANBAZ, N. T. KIRKLAND, E. STACE, T. WOODFIELD, M. P. STAIGER u. G. J. DIAS (2012): Magnesium alloys: predicting in vivo corrosion with in vitro immersion testing. J. Biomed. Mater. Res. B Appl. Biomater. 100, 1134-1141 WALKER, J., S. SHADANBAZ, T. B. F. WOODFIELD, M. P. STAIGER u. G. J. DIAS (2014a): Magnesium biomaterials for orthopedic application: A review from a biological perspective. J. Biomed. Mater. Res. B Appl. Biomater. 102, 1316-1331 WALKER, S. SHADANBAZ, T. B. F. WOODFIELD, M. P. STAIGER u. G. J. DIAS (2014b): The in vitro and in vivo evaluation of the biocompatibility of Mg alloys. Biomed Mater 9, 15006 WALTON J. R. (2014): Chronic Aluminum Intake Causes Alzheimer's Disease: Applying Sir Austin Bradford Hill's Causality Criteria. J. Alzheimers Dis. 40, 765-838 WANG Y. (1997): Beitrag zur Verbesserung korrosiver Eigenschaften von superleichten Magnesium-Lithium-Basislegierungen. Düsseldorf, Univ, Diss. WANG, H., S. GUAN, Y. WANG, H. LIU, H. WANG, L. WANG, C. REN, S. ZHU u. K. CHEN (2011): In vivo degradation behavior of Ca-deficient hydroxyapatite coated Mg-Zn-Ca alloy for bone implant application. Colloids Surf B Biointerfaces 88, 254-259 WANG, B., P. HUANG, C. OU, K. LI, B. YAN u. W. LU (2013): In vitro corrosion and cytocompatibility of ZK60 magnesium alloy coated with hydroxyapatite by a simple chemical conversion process for orthopedic applications.
74
Int J Mol Sci 14, 23614-23628 WANG, X. P., X.-L. ZHANG, Z.-G. LI u. X.-G. YU (2005): A first order system model of fracture healing. J Zhejiang Univ Sci B 6B, 926-930 WEHNER, T., U. WOLFRAM, T. HENZLER, F. NIEMEYER, L. CLAES u. U. SIMON (2010): Internal forces and moments in the femur of the rat during gait. J Biomech 43, 2473-2479 WEIZBAUER, A., J.-M. SEITZ, P. WERLE, J. HEGERMANN, E. WILLBOLD, R. EIFLER, H. WINDHAGEN, J. REIFENRATH u. H. WAIZY (2014): Novel magnesium alloy Mg-2La caused no cytotoxic effects on cells in physiological conditions. Mater Sci Eng C Mater Biol Appl 41, 267-273 WILLBOLD, E., K. KALLA, I. BARTSCH, K. BOBE, M. BRAUNEIS, S. REMENNIK, D. SHECHTMAN, J. NELLESEN, W. TILLMANN, C. VOGT u. F. WITTE (2013): Biocompatibility of rapid-solidified magnesium alloy RS66 as a temporary biodegradable metal. Acta Biomater 9, 8509-8517 WILLBOLD, E., A. A. KAYA, A. R. KAYA u. F. W. BECKMANN (2011): corrosion of magnesium alloy AZ31 screws is dependent on the implantation site. Mater Sci Eng B, 1835-1840 WILLHITE, C. C., G. L. BALL u. C. J. MCLELLAN (2012): Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water. Crit. Rev. Toxicol. 42, 358-442 WINDHAGEN, H., K. RADTKE, A. WEIZBAUER, J. DIEKMANN, Y. NOLL, U. KREIMEYER, R. SCHAVAN, C. STUKENBORG-COLSMAN u. H. WAIZY (2013): Biodegradable magnesium-based screw clinically equivalent to titanium screw in hallux valgus surgery: short term results of the first prospective, randomized, controlled clinical pilot study. Biomed Eng Online 12, 62 WITTE, F. (2010): The history of biodegradable magnesium implants: a review. Acta Biomater 6, 1680-1692 WITTE, F., I. ABELN, E. SWITZER, V. KAESE, A. MEYER-LINDENBERG u. H. WINDHAGEN (2007a): Evaluation of the skin sensitizing potential of biodegradable magnesium alloys. J Biomed Mater Res A 86, 1041-1047 WITTE, F., F. FEYERABEND, P. MAIER, J. FISCHER, M. STÖRMER, C. BLAWERT, W. DIETZEL u. N. HORT (2007b): Biodegradable magnesium-hydroxyapatite metal matrix composites.
75
Biomaterials 28, 2163-2174 WITTE, F., J. FISCHER, J. NELLESEN, H. A. CROSTACK, V. KAESE, A. PISCH, F. BECKMANN u. H. WINDHAGEN (2006): In vitro and in vivo corrosion measurements of magnesium alloys. Biomaterials 27, 1013-1018 WITTE, F., J. FISCHER, J. NELLESEN, C. VOGT, J. VOGT, T. DONATH u. F. BECKMANN (2010): In vivo corrosion and corrosion protection of magnesium alloy LAE442. Acta Biomater 6, 1792-1799 WITTE, F., V. KAESE, H. HAFERKAMP, E. SWITZER, A. MEYER-LINDENBERG, C. J. WIRTH u. H. WINDHAGEN (2005): In vivo corrosion of four magnesium alloys and the associated bone response. Biomaterials 26, 3557-3563 WITTE, F., H. ULRICH, C. PALM u. E. WILLBOLD (2007c): Biodegradable magnesium scaffolds: Part II: Peri-implant bone remodeling. J Biomed Mater Res A 81, 757-765 WITTE, F., H. ULRICH, M. RUDERT u. E. WILLBOLD (2007d): Biodegradable magnesium scaffolds: Part 1: appropriate inflammatory response. J Biomed Mater Res A 81, 748-756 WOLF F. I. u. A. CITTADINI (2003): Chemistry and biochemistry of magnesium. Mol Aspects Med 24, 3-9 WONG, H. M., K. W. K. YEUNG, K. O. LAM, V. TAM, P. K. CHU, K. D. K. LUK u. K. M. C. CHEUNG (2010): A biodegradable polymer-based coating to control the performance of magnesium alloy orthopaedic implants. Biomaterials 31, 2084-2096 WORLD HEALTH ORGANISATION (2007): Evaluation of certain food additives and contaminants WORLD HEALTH ORGANIZATION Geneva, Switzerland WHO Press XU, L., F. PAN, G. YU, L. YANG, E. ZHANG u. K. YANG (2009): In vitro and in vivo evaluation of the surface bioactivity of a calcium phosphate coated magnesium alloy. Biomaterials 30, 1512-1523 XU, L., E. ZHANG, D. YIN, S. ZENG u. K. YANG (2008): In vitro corrosion behaviour of Mg alloys in a phosphate buffered solution for bone implant application. J Mater Sci Mater Med 19, 1017-1025
76
YANG, L., Y. HUANG, F. FEYERABEND, R. WILLUMEIT, C. MENDIS, K. U. KAINER u. N. HORT (2013): Microstructure, mechanical and corrosion properties of Mg-Dy-Gd-Zr alloys for medical applications. Acta Biomater 9, 8499-8508 YU, K., L. CHEN, J. ZHAO, S. LI, Y. DAI, Q. HUANG u. Z. YU (2012): In vitro corrosion behavior and in vivo biodegradation of biomedical β-Ca3(PO4)2/Mg–Zn composites. Acta Biomater 8, 2845-2855 YUEN C. K. u. W. Y. IP (2010): Theoretical risk assessment of magnesium alloys as degradable biomedical implants. Acta Biomater 6, 1808-1812 ZBERG, B., P. J. UGGOWITZER u. J. F. LÖFFLER (2009): MgZnCa glasses without clinically observable hydrogen evolution for biodegradable implants. Nat Mater 8, 887-891 ZHANG, E., L. XU, G. YU, F. PAN u. K. YANG (2009): In vivo evaluation of biodegradable magnesium alloy bone implant in the first 6 months implantation. J Biomed Mater Res A 90, 882-893 ZHANG, S., X. ZHANG, C. ZHAO, J. LI, Y. SONG, C. XIE, H. TAO, Y. ZHANG, Y. HE, Y. JIANG u. Y. BIAN (2010): Research on an Mg-Zn alloy as a degradable biomaterial. Acta Biomater 6, 626-640 ZHENG, Y. F., X. GU u. F. WITTE (2014): Biodegradable metals. Mat Sci Eng R Reports 77, 1-34 ZREIQAT, H., C. R. HOWLETT, A. ZANNETTINO, P. EVANS, G. SCHULZE-TANZIL, C. KNABE u. M. SHAKIBAEI (2002): Mechanisms of magnesium-stimulated adhesion of osteoblastic cells to commonly used orthopaedic implants. J Biomed Mater Res 62, 175-184
77
8. Presentation of the own work
I. Reifenrath, J., Krause, A., Bormann, D., von Rechenberg, B., Windhagen, H.,
Meyer-Lindenberg, A.: Profound differences in biocompatibility of two very similar
Rare-earth containing Mg-alloys, Mat.- Wiss- u.Werkstofftech. 2010, 41, 12, p. 1054–
1061, doi: 10.1002/mawe.201000709
Idea, study design: Meyer-Lindenberg, Windhagen, Bormann
Study performance: Krause, Meyer-Lindenberg
Evaluation, discussion: Reifenrath, von Rechenberg
Manuscript preparation: Reifenrath
Corresponding author: Reifenrath
II. Erdmann, N., Angrisani, N., Reifenrath, J., Lucas, A., Thorey, F., Bormann, D.,
Meyer-Lindenberg A.: Biomechanical testing and degradation analysis of MgCa0.8
alloy screws: A comparative in vivo study in rabbits, Acta Biomater., 2010, 7, 3, p.
1421-1428 doi:10.1016/j.actbio.2010.10.03
Idea, study design: Meyer-Lindenberg, Reifenrath, Thorey, Bormann
Study performance: Erdmann, Reifenrath, Angrisani, Meyer-Lindenberg, Lucas
Evaluation, discussion: Erdmann
Manuscript preparation: Erdmann
Corresponding author: Erdmann
III. Erdmann, N., Bondarenko, A., Hewicker-Trautwein, M., Angrisani N., Reifenrath,
J., Lucas, A., Meyer-Lindenberg, A.: Evaluation of the soft tissue biocompatibility of
MgCa0.8 and surgical steel 316L in vivo: a comparative study in rabbits, Biomed.
Eng. Online, 2010, 9, 63
78
Idea, study design: Meyer-Lindenberg, Reifenrath
Study performance: Erdmann, Reifenrath, Angrisani, Meyer-Lindenberg,
Lucas, Bondarenko
Evaluation, discussion: Erdmann, Bondarenko, Hewicker-Trautwein
Manuscript preparation: Erdmann, Angrisani
Corresponding author: Erdmann
IV. Badar, M., Reifenrath, J., Rittershaus, D., Seitz, J.-M., Bormann, D., Bach, F-W.,
Hauser, H., Meyer-Lindenberg, A., Mueller, P.P.: In vitro and in vivo models for the
molecular evaluation of cellular responses to magnesium, Biomed Tech 2010, 55,
Suppl. 1, doi: 10.1515/BMT.2010.125
Idea, study design: Meyer-Lindenberg, Reifenrath, Bach, Hauser, Mueller
Study performance: Badar, Mueller, Seitz, Bormann, Reifenrath
Evaluation, discussion: Badar, Reifenrath, Rittershaus
Manuscript preparation: Badar, Reifenrath, Mueller
Corresponding author: Badar
V. Reifenrath, J., Bormann, D., Meyer-Lindenberg, A.: Magnesium alloys as
promising degradable implant materials in orthopaedic research; Chapter 6 in
Magnesium alloys – corrosion and surface treatments; Czerwinski F, Rijeka Intech,
2011, p. 93-108, ISBN 978-953-307-972-1
Idea, design: Meyer-Lindenberg, Reifenrath, Bormann
Study performance: Reifenrath
Evaluation, discussion: Reifenrath
Manuscript preparation: Reifenrath
Corresponding author: Reifenrath
79
VI. Reifenrath, J., Gottschalk, D., Angrisani, N., Besdo, S., Meyer-Lindenberg, A.:
Axial forces and bending moments in the loaded rabbit tibia in vivo; Acta Vet. Scand.
2012, 54, 21, doi:10.1186/1751-0147-54-21
Idea, study design: Reifenrath, Meyer-Lindenberg, Besdo
Study performance: Reifenrath, Gottschalk, Angrisani, Meyer-Lindenberg
Evaluation, discussion: Reifenrath, Besdo, Gottschalk
Manuscript preparation: Reifenrath, Besdo
Corresponding author: Reifenrath
VII. Hampp, C., Ullmann, B., Reifenrath, J., Angrisani, N., Dziuba, D., Bormann, D.,
Seitz, J.-M., Meyer-Lindenberg, A.: Research on the Biocompatibility of the New
Magnesium Alloy LANd442 – An In Vivo Study in the Rabbit Tibia over 26 Weeks;
Adv. Eng. Mater. 2011, 14, 3, B28-B37, doi: 10.1002/adem.201180066
Idea, study design: Meyer-Lindenberg, Bormann
Study performance: Ullmann, Hampp, Angrisani, Dziuba, Seitz
Evaluation, discussion: Hampp, Reifenrath, Angrisani
Manuscript preparation: Hampp
Corresponding author: Hampp
VIII. Ullmann, B., Reifenrath, J., Dziuba, D., Seitz, J.-M., Bormann, D., Meyer-
Lindenberg, A.: In Vivo Degradation Behavior of the Magnesium Alloy LANd442 in
Rabbit Tibiae; Materials 2011, 4, p. 2197-218; doi: 10.3390/ma4122197
Idea, study design: Meyer-Lindenberg, Bormann
Study performance: Ullmann, Reifenrath, Dziuba, Seitz, Meyer-Lindenberg
Evaluation, discussion: Ullmann
80
Manuscript preparation: Ullmann, Dziuba, Reifenrath
Corresponding author: Ullmann
IX. Huehnerschulte, T. A., Reifenrath, J., von Rechenberg, B., Dziuba, D., Seitz, J.
M., Bormann, D., Windhagen, H., Meyer-Lindenberg A.: In vivo assessment of the
host reactions to the biodegradation of the two novel magnesium alloys ZEK100 and
AX30 in an animal model, Biomed. Eng. Online, 2012, 11, 14
Idea, study design: Meyer-Lindenberg, Reifenrath, Windhagen,
Study performance: Huehnerschulte, Reifenrath, Dziuba, Seitz, Bormann
Evaluation, discussion: Huehnerschulte, von Rechenberg
Manuscript preparation: Huehnerschulte, Dziuba
Corresponding author: Reifenrath
X. Hampp, C., Angrisani, N., Reifenrath, J., Bormann, D., Seitz, J.-M., Meyer-
Lindenberg, A.: Evaluation of the biocompatibility of two magnesium alloys as
degradable implant materials in comparison to titanium as non-resorbable material in
the rabbit, Mater. Sci. Eng. C, 2013, 33, p. 317-26,
doi.org/10.1016/j.msec.2012.08.046
Idea, study design: Meyer-Lindenberg, Reifenrath, Angrisani
Study performance: Hampp, Angrisani, Reifenrath, Meyer-Lindenberg, Seitz,
Bormann
Evaluation, discussion: Hampp, Reifenrath, Angrisani
Manuscript preparation: Hammp
Corresponding author: Hammp
81
XI. Reifenrath, J., Badar M., Dziuba, D., Müller, P. P., Heidenblut, T., Bondarenko, A.,
Meyer-Lindenberg, A.: Evaluation of cellular reactions to magnesium as implant
material in comparison to titanium and to glyconate using the mouse tail model, J.
Appl. Biomater. Funct. Mater., 2013, 11, 2, e89-94, doi: 10.5301/JABFM.5000150.
Idea, study design: Meyer-Lindenberg, Reifenrath, Mueller, Bormann
Study performance: Reifenrath, Badar, Dziuba, Heidenblut
Evaluation, discussion: Reifenrath
Manuscript preparation: Reifenrath
Corresponding author: Reifenrath
XII. Ullmann, B., Angrisani, N., Reifenrath, J., Seitz, J.M., Bormann, D., Bach, F.W.,
Meyer-Lindenberg, A.: The effects of handling and storage on magnesium based
implants--first results, Mater. Sci. Eng. C Mater Biol. Appl., 2013, 33, 5, p. 3010-7,
doi: 10.1016/j.msec.2013.03.034.
Idea, study design: Meyer-Lindenberg, Bormann, Bach, Reifenrath, Angrisani
Study performance: Ullmann, Reifenrath, Angrisani, Seitz, Meyer-Lindenberg
Evaluation, discussion: Ullmann, Reifenrath, Seitz
Manuscript preparation: Ullmann, Reifenrath
Corresponding author: Ullmann
XIII. Ullmann, B., Reifenrath, J., Seitz, J.-M., Bormann, D., Meyer-Lindenberg, A.:
Influence of the grain size on the in vivo degradation behaviour of the magnesium
alloy LAE442, Proc. Inst. Mech. Eng. H J. Eng. Med., 2013, 27, 3, doi
10.1177/0954411912471495
Idea, study design: Meyer-Lindenberg, Bormann, Reifenrath, Angrisani
Study performance: Ullmann, Reifenrath, Angrisani, Meyer-Lindenberg
82
Evaluation, discussion: Ullmann, Seitz
Manuscript preparation: Ullmann, Reifenrath
Corresponding author: Reifenrath
XIV. Bondarenko, A., Angrisani, N., Meyer-Lindenberg, A., Seitz, J.M., Waizy, H.,
Reifenrath, J.: Magnesium-based bone implants: Immunohistochemical analysis of
peri-implant osteogenesis by evaluation of osteopontin and osteocalcin expression. J
Biomed. Mater. Res. A., 2013, 102, 5, p. 1449–57, doi: 10.1002/jbm.a.34828.
Idea, study design: Reifenrath, Meyer-Lindenberg, Waizy, Angrisani
Study performance: Bondarenko, Reifenrath, Angrisani, Seitz
Evaluation, discussion: Bondarenko
Manuscript preparation: Bondarenko, Angrisani, Reifenrath
Corresponding author: Angrisani
XV. Dziuba, D., Meyer-Lindenberg, A., Seitz, J. M., Waizy, H., Angrisani, N.,
Reifenrath, J.: Long-term in vivo degradation behaviour and biocompatibility of the
magnesium alloy ZEK100 for use as biodegradable bone implant; Acta Biomater.,
2013, 9, 10, p. 8548-60, doi.org/10.1016/j.actbio.2012.08.028
Idea, study design: Reifenrath, Meyer-Lindenberg, Waizy, Angrisani
Study performance: Dziuba, Angrisani, Reifenrath, Meyer-Lindenberg
Evaluation, discussion: Dziuba
Manuscript preparation: Dziuba
Corresponding author: Dziuba
XVI. Reifenrath, J., Angrisani, N., Erdmann, N., Lucas, A., Waizy, H., Seitz, J.M.,
Bondarenko, A., Meyer-Lindenberg, A.: Degrading magnesium screws ZEK100:
83
biomechanical testing, degradation analysis and soft-tissue biocompatibility in a
rabbit model. Biomed. Mater., 2013, 8, 4, p. 045012, doi: 10.1088/1748-
6041/8/4/045012.
Idea, study design: Meyer-Lindenberg, Reifenrath, Angrisani, Lucas
Study performance: Erdmann, Reifenrath, Angrisani
Evaluation, discussion: Reifenrath, Angrisani
Manuscript preparation: Reifenrath
Corresponding author: Reifenrath
XVII. Weizbauer, A., Modrejewski, C., Behrens, S., Klein, H., Helmecke, P., Seitz,
J.M., Windhagen, H., Möhwald, K., Reifenrath, J., Waizy,H.,: Comparative in vitro
study and biomechanical testing of two different magnesium alloys, Biomater. Appl. J
Biomater Appl., 2014, 28, 8, p. 1264-73, doi: 10.1177/0885328213506758.
Idea, study design: Waizy, Windhagen, Reifenrath, Helmecke, Seitz
Study performance: Weizbauer, Modrejewski, Behrens, Klein, Helmecke, Seitz
Evaluation, discussion: Weizbauer
Manuscript preparation: Weizbauer
Corresponding author: Weizbauer
XVIII. Wolters, L., Angrisani, N., Seitz, J., Helmecke, P., Weizbauer, A., Reifenrath J.:
Applicability of Degradable Magnesium LAE442 Alloy Plate-Screw-Systems in a
Rabbit Model. Biomed. Tech., 2013, p. 227 doi:pii: /j/bmte.2013.58.issue-s1-C/bmt-
2013-4059/bmt-2013-4059.xml. 10.1515/bmt-2013-4059.
Idea, study design: Reifenrath, Angrisani, Helmecke, Weizbauer
Study performance: Wolters, Reifenrath, Angrisani
Evaluation, discussion: Wolters
84
Manuscript preparation: Wolters
Corresponding author: Wolters
XIX. Reifenrath, J., Roessig, C., Wolters, L., Seitz, J.-M., Helmecke, P., Angrisani, N.:
Implant location strongly influences degradation and applicability of magnesium
alloys for orthopaedic application, Europ. Cells Mat., 2013, 26, Suppl. 5, p.17, ISSN
1473-2262
Idea, study design: Reifenrath, Angrisani
Study performance: Roessig, Wolters, Reifenrath, Angrisani, Helmecke, Seitz
Evaluation, discussion: Reifenrath
Manuscript preparation: Reifenrath
Corresponding author: Reifenrath
XX: Reifenrath, J., Angrisani, N., Lalk, M., Besdo, S.: Replacement, refinement and
reduction: necessity of standardization and computational models for long bone
fracture repair in animals, J Biomed. Mater. Res. A., 2014, 102, 8, p. 2884-900
Idea, study design: Reifenrath, Angrisani, Besdo
Study performance: Reifenrath, Besdo
Evaluation, discussion: Reifenrath, Angrisani, Besdo
Manuscript preparation: Reifenrath, Besdo
Corresponding author: Reifenrath
XXI. Rössig, C., Angrisani, N., Besdo, S., Damm, N.B., Badenhop, M., Fedchenko,
N.,Helmecke, P., Seitz, J.M., Meyer-Lindenberg, A., Reifenrath, J.: Magnesium-
based intramedullary nailing system in a sheep model: Biomechanic evaluation and
85
first in vivo results, J. Vet. Sci. Med. Diagn. 2014, 4, 1, doi:10.4172/2325-
9590.1000150
Idea, study design: Reifenrath, Angrisani, Meyer-Lindenberg, Besdo, Seitz,
Helmecke
Study performance: Roessig, Helmecke, Damm, Badenhop, Reifenrath,
Angrisani
Evaluation, discussion: Roessig, Damm, Badenhop, Fedchenko
Manuscript preparation: Roessig
Corresponding author: Roessig
XII. Bracht, K., Angrisani, N., Seitz, J.M., Eifler, R., Weizbauer, A., Reifenrath, J.: The
influence of storage and heat treatment on a magnesium-based implant material: an
in vitro and in vivo study, Biomed Eng Online. 2015, 14, 92, doi: 10.1186/s12938-
015-0091-8.
Idea, study design: Reifenrath, Angrisani, Seitz, Weizbauer
Study performance: Bracht, Reifenrath, Angrisani, Eifler
Evaluation, discussion: Bracht
Manuscript preparation: Bracht, Angrisani, Reifenrath
Corresponding author: Angrisani
XIII. Wolters, L., Besdo, S., Angrisani, N., Wriggers, P., Hering, B., Seitz, J.M.,
Reifenrath, J.: Degradation behaviour of LAE442-based plate-screw-systems in an in
vitro bone model, J Mat. Sci. Eng. C, 2015, 49, p. 305–15
Idea, study design: Reifenrath, Angrisani, Seitz, Wriggers, Hering, Wolters
Study performance: Wolters, Hering
Evaluation, discussion: Wolters
86
Manuscript preparation: Wolters, Reifenrath
Corresponding author: Reifenrath
XIV. Rössig, C., Angrisani, N., Helmecke, P., Besdo, S., Seitz, J.M., Welke,
B.,Fedchenko, N., Kock, H., Reifenrath, J.: In vivo evaluation of a magnesium-based
degradable intramedullary nailing system in a sheep model, Acta Biomater. 2015, 25,
p. 369-83, doi: 10.1016/j.actbio.2015.07.025 16.03.2015
Idea, study design: Reifenrath, Angrisani, Besdo
Study performance: Roessig, Welke, Helmecke, Seitz, Fedchenko, Kock
Evaluation, discussion: Roessig, Welke, Kock, Fedchenko
Manuscript preparation: Roessig
Corresponding author: Roessig
87
9. Acknowledgement
First of all, I thank my husband Dr. Hans Reifenrath and my mother Margrit Hepke for
supporting my work in particular through taking care for our children Cora, Henrike
and Jette. Additionally I thank the former head of our research group Prof. Andrea
Meyer-Lindenberg for transferring me the group leadership. I thank all my colleges at
the Veterinary University, the Leibniz University and the Hannover Medical School
who were involved in all the interdisciplinary studies. My special thanks go to my
colleague Dr. Nina Angrisani, who helped me in planning, performing, evaluating and
publishing the scientific research. At least I thank the German Research Foundation
(DFG) for funding most of the studies within the collaborative research center SFB
599 (subprojects R2, R4 and R6).
top related