In vivo evaluation of degradable magnesium alloys as ... · protection by the use of high purity magnesium, alloying elements or coatings was very rare and might have been the reason

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)

http://www.aphorismen.de/zitat/16750

http://www.aphorismen.de/zitat/16750

http://www.aphorismen.de/autoren/person/841/Lord+George+Gordon+Noel+Byron

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.,



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




2011, 93-108, ISBN 978-953-307-972-1



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




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





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.


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



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




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




1421-1428, doi:10.1016/j.actbio.2010.10.03



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.



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


1473-2262


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


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


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




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

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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.

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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

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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

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8. Presentation of the own work

I. Reifenrath, J., Krause, A., Bormann, D., von Rechenberg, B., Windhagen, H.,



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




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




2011, p. 93-108, ISBN 978-953-307-972-1

Idea, design: Meyer-Lindenberg, Reifenrath, Bormann

Study performance: Reifenrath

Evaluation, discussion: Reifenrath



79


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




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




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






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




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


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




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




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



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





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


1473-2262

Idea, study design: Reifenrath, Angrisani

Study performance: Roessig, Wolters, Reifenrath, Angrisani, Helmecke, Seitz




XX: Reifenrath, J., Angrisani, N., Lalk, M., Besdo, S.: Replacement, refinement and



Idea, study design: Reifenrath, Angrisani, Besdo

Study performance: Reifenrath, Besdo

Evaluation, discussion: Reifenrath, Angrisani, Besdo

Manuscript preparation: Reifenrath, Besdo



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


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


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

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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).

Documents

In vivo evaluation of degradable magnesium alloys as ... · protection by the use of high purity magnesium, alloying elements or coatings was very rare and might have been the reason