Degradation und Biokompatibilität der neuen Magnesiumlegierung LANd442 im Vergleich zu LAE442 und der nicht degradablen Titanlegierung Ti6Al4V-Eli
nach intramedullärer Implantation in die Kaninchentibia
INAUGURAL-DISSERTATION
zur Erlangung des Grades einer Doktorin der Veterinärmedizin - Doctor medicinae veterinariae -
(Dr. med. vet.)
vorgelegt von Carolin Hampp Heilbronn – Neckargartach
Hannover 2012
Wissenschaftliche Betreuung: Univ.-Prof. Dr. med. vet. Andrea Meyer-Lindenberg Klinik für Kleintiere
Jetzt: Chirurgische und Gynäkologische Kleintierklinik der Ludwig-Maximilians-Universität München
1. Gutachterin: Univ.-Prof. Dr. med. vet. Andrea Meyer-Lindenberg 2. Gutachter: Univ.-Prof. Dr. med. vet. Ingo Nolte
Tag der mündlichen Prüfung: 10.05.2012
Diese Dissertation entstand im Rahmen des Sonderforschungsbereichs 599 „Zukunftsfähige bioresorbierbare und permanente Implantate aus metallischen und keramischen Werkstoffen“ im Teilprojekt R6 „Degradable Implantate“, gefördert durch die Deutsche Forschungsgemeinschaft (DFG).
Für meine Familie
Ergebnisse dieser Dissertation wurden in international anerkannten Fachzeitschriften mit Gutachtersystem (peer review) zur Veröffentlichung angenommen oder eingereicht:
• Advanced Engineering Materials: Advanced Biomaterials (angenommen am 29.10.2011)
DOI: 10.1002/adem.201180066
Research on the biocompatibility of the new magnesium alloy LANd442 – an in vivo study in the rabbit tibia over 26 weeks
C. Hampp, B. Ullmann, J. Reifenrath, N. Angrisani, D. Dziuba, D. Bormann, J.-M. Seitz, A. Meyer-Lindenberg
• Materials Science and Engineering: C (eingereicht am 07.03.2012)
Evaluation of the biocompatibility of two magnesium alloys as degradable implant materials in comparison to titanium as non resorbable material in the rabbit
C. Hampp, N. Angrisani, J. Reifenrath, D. Bormann, J.-M. Seitz, A. Meyer-Lindenberg
Teilergebnisse dieser Dissertation wurden auf folgenden Fachkongressen präsentiert:
• Euro BioMat 2011 – European Symposium on Biomaterials and related areas, Jena, 13.-14.04.2011
In-vivo research on the biocompatibility of the new magnesium alloy LANd442 on the basis of imaging procedures
C. Hampp, D. Rittershaus, J. Reifenrath, D. Bormann, J. Seitz, A. Meyer-Lindenberg
• Jahrestagung der Deutschen Gesellschaft für Biomaterialien, Gießen, 10.-12.11.2011; BioMaterialien, 12, 1-4, 2011.
Untersuchung der Biokompatibilität von degradablen Magnesiumlegierungen im Vergleich zu Titan im Kaninchenmodell
C. Hampp, J. Reifenrath, N. Angrisani, D. Bormann, J.-M. Seitz, A. Meyer-Lindenberg
Inhaltsverzeichnis
1 Einleitung ... 9
2 Publikation I ... 12
2.1 Abstract ...13
2.2 Introduction...13
2.3 Materials and Methods ...15
2.3.1 Implants...15
2.3.2 Animal model...15
2.3.3 Clinical investigation...16
2.3.4 Radiological investigation ...16
2.3.5 In vivo µ-computed tomography ...16
2.3.6 Intravital staining for histologically investigating new bone growths ...17
2.3.7 Euthanasia ...18
2.3.8 Ex vivo µ-computed tomography ...18
2.3.9 Histological investigations ...19
2.3.10 Statistics...20
2.4 Results...21
2.4.1 Clinical investigation...21
2.4.2 Radiological investigations ...21
2.4.3 In vivo µ-computed tomography ...22
2.4.4 Ex vivo µ-computed tomography ...25
2.4.5 Histological investigations ...27
2.5 Discussion...29
2.6 Conclusions ...37
2.7 Acknowledgements ...38
2.8 References ...39
3 Publikation II ... 45
3.1 Abstract ...46
4 Diskussion ... 47
5 Zusammenfassung... 62
6 Summary ... 65
7 Literaturverzeichnis ... 68
1 Einleitung
In der Orthopädie ist eine der wichtigsten Fragestellungen die nach den am besten geeigneten Osteosynthesematerialien. Seit Jahren wird neben den etablierten Materialien wie Titan und Stahl, die sich durch eine hohe Stabilität auszeichnen, an resorbierbaren Implantatmaterialien geforscht. Bereits etabliert sind hierbei verschiedene Kunststoffe wie PLA, PGA und PMMA (HOFMANN et al. 1990;
GRIFFITH 2000). Diese sind jedoch nicht stabil genug, um Anwendung am belasteten Knochen zu finden (SONG 2007; SEALY a. GUO 2010; LICHTE et al.
2011). Daher konzentriert sich die Forschung auf degradable Metalle, wobei Magnesium im Fokus dieser neuen Orientierung steht (WITTE et al. 2004; STAIGER et al. 2006; FEYERABEND et al. 2010; MEYER-LINDENBERG et al. 2010;
HUEHNERSCHULTE et al. 2011). Das Leichtmetall Magnesium zeichnet sich durch einen Elastizitätsmodul von 45 GPa aus (AVEDESIAN 1999), der im Vergleich zu Titan (105-110 GPa (LONG a. RACK 1998)) oder Stahl (190-210 GPa (LEVESQUE et al. 2004; MOAVENI 2010)) eine weitaus geringere Differenz zu dem des kortikalen Knochens (18,6 GPa (RHO et al. 1993)) aufweist. Beachtet man zusätzlich die Tatsache, dass ein Osteosyntheseimplantat zunächst die auftretenden Belastungen aushalten muss, um den regenerierenden Knochen vor einer erneuten Fraktur zu bewahren, erscheint ein etwas höherer Elastizitätsmodul durchaus vorteilhaft. Ist der Unterschied jedoch zu groß, kommt es zum Phänomen des stress-shielding (SEALY a. GUO 2010), bei dem das Implantat den heilenden Knochen von jeglicher Belastung abschirmt. Hierdurch kommt es zu einer verminderten Mineralisierung des Knochens (TONINO et al. 1976; BENLI et al. 2008) und die Gefahr der Refrakturierung nach Entfernen des Implantates steigt, da das Implantatlager zunächst wieder vom Knochen stabilisiert werden muss (WITTE et al. 2004).
Herkömmliche Implantatmaterialien haben zusätzlich den Nachteil, dass sie eine Fremdkörperreaktion hervorrufen können, wenn sie im Körper belassen werden (VOGGENREITER et al. 2003; LICHTE et al. 2011). Um diese Probleme zu umgehen, werden die Implantate häufig entfernt, nachdem der erwünschte Heilungseffekt eingetreten ist. Eine solche Zweitoperation zur Entfernung der eingebrachten Implantate ist bei einem degradablen Osteosynthesematerial hinfällig
und stellt dadurch einen weiteren Vorteil von Magnesiumlegierungen dar (SONG 2007; BENLI et al. 2008). Da jede Operation Kosten, Schmerzen sowie ein nie auszuschließendes Narkoserisiko für den Patienten bedeutet (ROKKANEN et al.
2000; WITTE et al. 2004), bietet ein degradierendes Implantat die Möglichkeit der Verringerung dieser Faktoren.
Zusätzlich zeichnet sich Magnesium als körpereigenes Element grundsätzlich durch eine sehr gute Verträglichkeit aus. Ein erhöhter Magnesiumspiegel, wie er im Rahmen des Abbaus eingebrachter Implantate eventuell entstehen könnte, wird durch den Körper selbst reguliert (VORMANN 2003) und hat in den zu erwartenden Höchstkonzentrationen keine toxischen Folgen (SARIS et al. 2000).
Jedoch kann Magnesium nicht als alleiniges Element für Osteosyntheseimplantate verwendet werden, da seine Primärstabilität nicht ausreicht, um den Knochen postoperativ optimal zu stützen (MCBRIDE 1938) und diese zudem in chloridhaltigen Medien zu schnell abnimmt (STAIGER et al. 2006; MEYER-LINDENBERG et al.
2010). Daher müssen andere Elemente zulegiert werden, um die Eigenschaften des Materials zu verbessern. Bisherige Studien konnten zeigen, dass sich Seltene Erden hierfür besonders eignen (WITTE et al. 2005; MINGXING et al. 2007; KRAUSE 2008;
HORT et al. 2009). In der Folge wurden verschiedene Legierungen wie LAE442, ZEK100 und WE43 entwickelt und sowohl in vitro als auch in vivo getestet, wobei sich LAE442 sehr vielversprechend zeigte (WITTE et al. 2005; THOMANN et al.
2009). Allen diesen Seltene Erden enthaltenden Materialien ist gemein, dass das Mischungsverhältnis der einzelnen Elemente innerhalb der Legierungen nicht genau bekannt ist.
Hierin liegt ein Problem für die Reproduzierbarkeit der Legierung und damit für die Gewährleistung einer gleich bleibenden Qualität, was eine essentielle Voraussetzung für den Einsatz in der Medizin darstellt. Daher wurde die neue Magnesiumlegierung LANd442 entwickelt (SEITZ et al. 2011), die aufgrund der bisherigen guten Ergebnisse auf LAE442 basiert (WITTE et al. 2005; THOMANN et al. 2009).
Verschiedene Studien stuften unter anderen Elementen der Seltenen Erden Neodym in in vitro Versuchen als gut verträglich ein (DRYNDA et al. 2009; FEYERABEND et
al. 2010). Daher wurde der Anteil des in LAE442 enthaltenen Seltenen Erden- Gemisches bei der Entwicklung der neuen Legierung durch das Einzelelement Neodym ersetzt, um eine genau definierte Legierungszusammensetzung zu erreichen (ROKHLIN 2003). Dieser Ansatz wurde bereits bei der Legierung LACer442 verfolgt, bei der die Seltene Erden-Mischung durch das Element Cer ersetzt wurde, die sich jedoch im in vivo Versuch als nicht biokompatibel herausstellte (REIFENRATH et al. 2010). Allerdings besitzt Neodym im Gegensatz zu Cer laut einer Verträglichkeitsstudie in vitro kein toxisches Potential (FEYERABEND et al. 2010) bzw. erst in sehr hohen Konzentrationen (DRYNDA et al. 2009), sodass von einer besseren Verträglichkeit ausgegangen wird.
In der zugänglichen Literatur existieren jedoch keine in vivo Studien, die die Biokompatibilität von Neodym als Legierungselement untersuchen. Daher war es das Ziel der vorliegenden Arbeit, die Biokompatibilität der neuen Legierung LANd442 in vivo unter Berücksichtigung der Degradation über verschiedene Zeiträume (vier und acht Wochen bzw. sechs Monate) im Kaninchenmodell zu prüfen. Als Vergleichsmaterial wurde die als vielversprechend geltende Magnesiumlegierung LAE442 herangezogen und das etablierte Osteosynthesematerial Titan diente als Kontrolle. Zudem wurden Tibiae in die Untersuchung einbezogen, bei denen die Operation durchgeführt wurde, die aber kein Implantat erhielten (Leertibiae), um die Auswirkungen der Operationsmethode von den potentiellen durch die Implantate induzierten Vorgängen am Knochen zu unterscheiden.
2 Publikation I
Das Manuskript wurde am 07.03.2012 im Journal „Materials Science and Engineering: C“ zur Veröffentlichung eingereicht.
Evaluation of the biocompatibility of two magnesium alloys as degradable implant materials in comparison to titanium as non
resorbable material in the rabbit
Carolin Hampp*, Nina Angrisani, Janin Reifenrath, Dirk Bormann, Jan-Marten Seitz, Andrea Meyer-Lindenberg
C. Hampp, Dr. N. Angrisani, Dr. J. Reifenrath:
Small Animal Clinic, University of Veterinary Medicine Hanover, Bünteweg 9, 30559 Hanover, Germany
Dr. D. Bormann, J.-M. Seitz:
Institute of Materials Science, Leibniz University Hanover, An der Universität 2, 30823 Garbsen, Germany
Prof. Dr. A. Meyer-Lindenberg:
Clinic for Small Animal Surgery and Reproduction, Centre of Clinical Veterinary Medicine, Faculty of Veterinary Medicine, Ludwig-Maximilians-University München, Veterinärstraße 13, 80539 Munich, Germany
* Corresponding author
2.1 Abstract
The aim of this study is to compare the biocompatibility of the two magnesium based alloys LAE442 and LANd442 with that of titanium. For this purpose, cylindrical implants were introduced into the medullary cavity of rabbit’s tibiae for 4 and 8 weeks. Animals without any implant served as a control. In the follow-up, clinical, X- ray and µCT-investigations were performed to evaluate the reactions of the bone towards the implanted materials. After euthanasia, ex vivo µCT- and histological investigations were performed to verify the results of the in vivo tests. It could be shown that all materials induce changes in the bone. Whereas LANd442 caused the most pronounced reactions, such as increasing bone volume and bone porosity and decreasing bone density, titanium showed the most bone-implant contact by forming trabeculae. The tibiae of rabbits without implants also reacted by forming cavities, it is therefore assumed that the surgery method itself influences the bone. Compared to LANd442, LAE442 seems to be the more qualified alloy since it demonstrated better clinical tolerance.
2.2 Introduction
The treatment of fractures is a major field within orthopaedic surgery. Commonly used materials are stainless steel or titanium (DISEGI a. ESCHBACH 2000;
POHLER 2000; FERRARIS et al. 2011) which have been long established and are still considered appropriate and almost unrivalled. This is evident due to the fact that current research focuses on the improvement of these established materials (JAIMES et al. 2010; KERÄNEN et al. 2011) rather than developing alternatives.
However, the disadvantage of stainless steel and titanium is that both materials are non resorbable. Therefore, they have to be removed, following complete bone healing, in a second surgery or, when left in the organism, can cause foreign body reactions (VOGGENREITER et al. 2003).
Competing with these durable materials are resorbable materials like polymers and ceramics which spare the patient a second surgical procedure to remove the implant.
However, polymers can also cause foreign body reactions and neither material has sufficient mechanical load carrying capacity for the use in weight bearing bones (LICHTE et al. 2011). In recent years, degradable implant materials which are based
on magnesium were studied to a greater extent (WITTE et al. 2005; REIFENRATH et al. 2010; ATRENS et al. 2011). They should combine the ability to degrade with sufficient mechanical properties. The advantages of employing magnesium as the main component are obvious: as an essential element of the human body, magnesium is well tolerated (SARIS et al. 2000) and it prevents stress shielding during bone healing (SEALY a. GUO 2010) due to its Young’s modulus which, of all the osteosynthetic materials, is closest to that of bone (STAIGER et al. 2006; XU et al. 2007). Several studies showed that the mechanical properties and the biocompatibility of magnesium can be influenced by alloying with other elements (STAIGER et al. 2006; KIM et al. 2008; ATRENS et al. 2011). Particularly the use of rare earth elements (RE) led to good results (MORDIKE 2002; MINGXING et al.
2007; HORT et al. 2009). The alloy LAE442 which, besides magnesium, lithium and aluminium, contains a mixture of various RE proved especially promising (THOMANN et al. 2009; KRAUSE et al. 2010; WITTE et al. 2010).
Previous studies of this alloy described implantation periods of three or more months (THOMANN et al. 2009; KRAUSE et al. 2010). However, literature on earlier time points, which would represent the remodelling processes during fracture healing, is currently lacking. For this reason, the present study chose implantation periods of 4 and 8 weeks to evaluate the potentially early occurrence of bone remodelling processes. Owing to the previous good results for LAE442, this alloy was included in the present study and was compared to LANd442, which is based on LAE442 but only contains neodymium instead of the RE mixture. The replacement of the RE mixture by the single element neodymium aims to achieve a better reproducibility. As control groups, two models were chosen. On the one hand, titanium implants were introduced; on the other hand, tibiae were used, which underwent the same surgical procedure but without receiving an implant as performed in previous studies (THOMANN et al. 2009; HUEHNERSCHULTE et al. 2011).
2.3 Materials and Methods 2.3.1 Implants
In this study, cylindrical implants (Ø 2.5 mm, 25 mm length) made of the magnesium based alloys LAE442 and LANd442 were used as well as titanium-alloy implants of the same geometry which were employed as a control (Ti6Al4V-Eli; S+D Spezialstahl Handelsgesellschaft mbH, Stelle, Germany). The magnesium implants were specially produced for this study according to a previously published method (KRAUSE et al.
2010; THOMANN et al. 2010a; HUEHNERSCHULTE et al. 2011; SEITZ et al. 2011).
Besides 90 wt% magnesium, the alloy LAE442 contains 4 wt% lithium, 4 wt%
aluminium and 2 wt% of a rare earth mixture. The alloy LANd442 is based on LAE442 and contains the same proportions of magnesium, lithium and aluminium.
Here however, the rare earth mixture was replaced by 2 wt% of the single element neodymium.
2.3.2 Animal model
The animal tests were approved by the Federal Office of Consumer Protection and Food Safety, according to paragraph 8 of the animal protection law, with the reference number 33.9-42502-04-07/1363.
For the current study 28 New Zealand White Rabbits (Charles River, Kisslegg, Germany) were randomly placed into six groups, a 4- and an 8-week group for each implant material (table 1). The implants of the LAE442- and LANd442-groups were introduced according to a previously described method (HAMPP et al. 2012) into the medullary cavity of both tibiae in four rabbits, respectively. Two rabbits only received one implant each. In their other hind leg, surgery was carried out using the same procedure, but no implant was inserted. The titanium implants were introduced into two rabbits per time group on both sides at the same location. After surgery, the rabbits received enrofloxacin (10 mg/kg, Baytril® 2.5%, Bayer HealthCare, Leverkusen, Germany) and meloxicam (0.15 mg/kg, Metacam®, Boehringer Ingelheim, Ingelheim, Germany) for a time period of ten days.
Table 1: Overview of the number of tibiae per implant material and time period material 4-week group 8-week group
LAE442 10 10
LANd442 10 10
Titanium 4 4
without implant 4 4
2.3.3 Clinical investigation
The animals were examined daily according to a previously described method (HAMPP et al. 2012). These examinations focused on possible changes occurring in the rabbits’ hind legs. If the rabbits showed lameness, they were treated with meloxicam (0.15 mg/kg, Metacam®, Boehringer Ingelheim, Ingelheim, Germany) beyond day 10 after surgery.
2.3.4 Radiological investigation
The tibiae of all rabbits were radiologically investigated once every week in two layers (anterior-posterior, mediolateral; 48 kV and 6.3 mAs) to evaluate bone alterations and the development of gas. All changes were assessed by a semi- quantitative scoring according to HUEHNERSCHULTE et al. (2011) which allows score values from 0 (no occurrence) to 3 (strong occurrence) for the individual parameters (growths at the implantation site at the proximal tibia, growths at the implant location at the diaphysis, gas, changes in the medullary cavity and corticalis).
Finally, all score values of the rabbits in one group were summed to a total score and the respective group’s mean value was computed for each time point of the investigation.
2.3.5 In vivo µ-computed tomography
Over the investigation periods of 4 and 8 weeks, µ-computed tomography evaluations (µCT) of the rabbits were performed (resolution: 41 µm, projections: 1000 at 0-180°, integration time: 100 ms; XtremeCT, Scanco Medical, Zurich, Switzerland).
The rabbits of the 4-week groups were scanned weekly and those in the time period
over 8 weeks, biweekly. The investigation was done under general anaesthesia. The evaluated area was limited by the knee joint space in the proximal direction and reached up to about 5 mm beneath the implant in the distal direction. The analysis of the µCT-investigations was done in both two and also three dimensions.
Two-dimensional evaluation
The overall impression of the bone including formation of cavities, periosteal and endosteal new bone growth, the growth behaviour onto the implant as well as the formation of gas bubbles were evaluated by an established semi-quantitative scoring (HUEHNERSCHULTE et al. 2011) based on nine selected cross-sections (THOMANN et al. 2010a). For every parameter, score values from 0 to 3 were given for each cross-section (no occurrence: value 0, strong occurrence: value 3) and a mean value per time group and per investigation time point was computed. The tibiae of the animals with titanium implants were not investigated in this way because of an expected insufficient resolution of detail as a consequence of the titanium’s stronger absorption of X-rays (BERNHARDT et al. 2004).
Three-dimensional evaluation
On the basis of every in vivo µCT-investigation a three-dimensional evaluation of each tibia was performed, including the bone area, in which the bone-implant- compound was completely visible. Using the computational software (µCT Evaluation Program V6.0; Scanco Medical, Zurich, Switzerland), the threshold for the evaluation of the bone was determined to be 160. Subsequently, the computations of the tibia areas were used to compute the bone volume (in mm³/slice), the bone density (in mg HA/ccm) as well as the bone porosity (in %). An evaluation of mean values and standard deviations was done for every investigation time point for every time group.
2.3.6 Intravital staining for histologically investigating new bone growths For the fluorescent-microscopic investigation following euthanasia, the animals received three different fluorochromes which were chosen according to RAHN (1976) and injected subcutaneously at specific times (table 2).
Table 2: Overview of the specific injection times of the fluorochromes and the resulting time periods calcein green xylenolorange tetracycline time period 1 time period 2 4-week groups day 3+6 day 13+16 day 23+26 day 3-13 day 16-23 8-week groups day 3+6 day 27+30 day 51+54 day 3-27 day 30-51
2.3.7 Euthanasia
At the end of the investigation time period the rabbits were painlessly euthanized as described in previous studies (LALK et al. 2010; HUEHNERSCHULTE et al. 2011;
HAMPP et al. 2012). The rabbit’s tibiae were removed and, after removal of adherent tissue, fixed in formaldehyde (4%). The left tibiae of the rabbits containing the magnesium implants were not used for the following investigations.
2.3.8 Ex vivo µ-computed tomography
After removing the tibiae, they were again evaluated by a µCT-investigation but using a higher resolution of 36 µm (integration time: 1 s; MicroCT80, Scanco Medical, Zurich, Switzerland). The evaluation of these µCT-scans corresponds to the two- dimensional evaluation of the in vivo scans but also includes the titanium implants (figure 1). In addition to this, an assessment of the implant degradation was performed by means of a semi-quantitative scoring for the examination of the implant’s cross-sections. Depending on the degradation rate (no degradation: value 0; initial changes at the implant’s edge: value 1; changes at the implant’s edge with crack formation: value 2; coarse structural changes: value 3) and subsequent to the score values determined for each implant (n = 9), the median, minimum and maximum values were computed for every group.
Figure 1: Ex vivo µCT cross-sections of the bone-implant-compounds of a) LAE442, b) LANd442, c) titanium, d) cross-section of a tibia without implant
2.3.9 Histological investigations
Following the µCT-investigations, the complete bone-implant-compound was detached and embedded in hydroxyethylmethacrylate (Technovit 7200 VLC, Heraeus Kulzer GmbH, Wehrheim, Germany) according to the manufacturer’s instructions. To produce the 50 µm thick histological cross-sections of the bone, the cutting and grinding technique was used according to DONATH (1988). At central cross-sections of the bone, fluorescent investigations were carried out as well as transmission microscopy investigations of histological slices stained with TRAP and toluidine blue.
Fluorescent-microscopic investigation
One cross-section per tibia was fluorescent-microscopically evaluated. Based on the intravitally injected fluorochromes, which were visible as double bands due to two successive injections, the mineral apposition rate (MAR) could be determined (PARFITT 1987). Therefore, the distance between the bands of two adjacent fluorochrome sequences was measured and divided by the number of days between the corresponding injections (PARFITT 1987). The time points of the intravital stainings resulted in two different time periods for the 4-week groups and the 8-week groups, respectively (table 2). In the present study, a multichannel image of the histological slices was generated using specific filters (FS 14, FS 18, FS 46, Carl Zeiss AG, Jena, Germany) and the distance at twelve specific locations was
measured according to an established method (REIFENRATH et al. 2011; HAMPP et al. 2012). Finally, mean values and standard deviations were computed for every slice to determine the mean value of the individual groups.
TRAP staining
One cross-section of each tibia was subjected to a TRAP staining (Tartrate-Resistant Acid Phosphatase; naphthol AS-MX phosphate / Fast Red TR Salt, Sigma Aldrich, St. Louis, USA) for viewing the osteoclasts (MOSTAFA et al. 1982; LINDUNGER et al. 1990). The counting of osteoclasts and Howship’s lacunae was carried out three times at x 200 magnification on every stained histological slice. Afterwards, mean values and standard deviations for all groups were determined.
Toluidine blue
The toluidine blue staining (0.1% toluidine blue O, Chroma, Münster, Germany) was applied to two histological cross-sections of the bone per tibia. Using a previously described semi-quantitative scoring (HAMPP et al. 2012), the slices were investigated at x 100 magnification for the following parameters: overall impression of the bone including cavities, periosteal bone growth and remodelling, endosteal bone growth and remodelling, bone-implant contact area, peri-implant fibrous capsule formation. For the evaluation, score values from 0 (no occurrence) to 3 (strong occurrence) were assigned depending on the parameter’s occurrence. Subsequent to this, the mean value as well as the standard deviation of all investigated cross- sections in one group was computed (LAE442, LANd442, without implant: n = 10;
titanium: n = 8).
2.3.10 Statistics
The values determined for the present study were analyzed using the Microsoft Office Excel program (Microsoft Office XP, Microsoft Corporation, Redmond, USA) and SPSS version 17.0 (SPSS: an IBM Company, Chicago, USA). Firstly, they were tested for a normal distribution. Normally distributed values were checked for statistical significance by means of a t-test or ANOVA, respectively; non-normally
distributed values were tested by means of Wilcoxon- or Mann-Whitney-tests.
Statistical significance existed for p < 0.05, whereas p < 0.01 indicated a highly significant difference.
2.4 Results
2.4.1 Clinical investigation
During the post-operative period, all rabbits showed swelling and coarse peripheral augmentation at the implantation's location. Redness occurred in 45 of 56 legs. Only one animal of the 4-week LAE442-group demonstrated wound dehiscence on day 15 and 16. Mild subcutaneous emphysema could be found in two legs of the 4-week LAE442-group (lasting 1 and 16 days, respectively) and four legs of the 8-week LANd442-group (1 to 7 days). Within this group two rabbits showed a low-grade lameness of one hind leg which lasted one day and resulted in the administration of meloxicam. No tibiae showed infections.
2.4.2 Radiological investigations
Within the 4-week groups, tibiae with magnesium implants showed initial alterations in week 1 (figure 2). Contrary to this, in the titanium-group an increase in the score could not be found before week 3, however, then an abrupt score value of 2.0 was found which increased further to the final value 2.5 in week 4.
The final value of the LAE442-group was also 2.5 whereas the 4-week LANd442 group reached a final score of 1.5. Also, tibiae without implants showed radiographic changes. The score values increased parallel to the LANd442-group from 0.3 in week 1 to 1.8 in week 4.
In contrast to this, all groups over 8 weeks showed initial changes in week 3. Here, in the titanium-group an increase to 1.0 score point was noted which, at the same time, appeared as the maximum final value obtained. The animals with magnesium implants showed a nearly parallel development, whereas the LAE442-group constantly showed higher values, but already attained the final value of 1.8 score points in week 7. The LANd442-group exhibited a stronger increase in week 8 to the
final value of 2.3 score points. The group without implants formed a plateau at 1.3 score points in week 5 to 7, but decreased finally to 0.7 score points.
The largest fraction of bone changes was contributed by the parameters for growth at the implantation site and growths at the diaphysis.
Figure 2: Development of the investigated bone alterations in all experimental groups by means of radiological investigations over a) 4 weeks, b) 8 weeks
2.4.3 In vivo µ-computed tomography Two-dimensional evaluation
In the two-dimensional evaluation of the in vivo µCT-scans, which excluded the titanium implants because of their expected irradiation, it was shown that not all parameters were equally pronounced. The highest score values were achieved for the formation of gas (figure 3 a), especially in the LANd442-group over 4 weeks. In contrast to this, gas was not even observed directly after surgery in either group without implants. This was followed by the formation of cavities (figure 3 b), mainly in the LANd442-group over 8 weeks (0.1 score point in week 8). Contrastingly, the group without implants showed less cavities (0.2 score points in week 8) in the corresponding time period. With 0.8 score points for their respective final time points, the LAE442-groups showed over 4 and 8 weeks the most periosteal formation of new bone which, in the groups without implants, was not detected over the entire investigation period (figure 3 c). However, the endosteal formation of new bone was most strongly pronounced in both LANd442-groups, while again both groups without implants showed the least changes (figure 3 d). All in all, less contact between bone
and implant existed (figure 3 e). Regarding this, most contacts were formed in the LAE442-group in week 6 and 8. No contact between bone and implant was detected at all in the LANd442-group over 8 weeks.
Figure 3: Development of the investigated parameters in all groups by means of two-dimensional evaluation of the in vivo µCT scans; a) formation of gas, b) formation of cavities, c) periosteal
formation of new bone, d) endosteal formation of new bone, e) bone-implant-contact
Three-dimensional evaluation
The three-dimensional evaluation resulted in different developments for the three investigated parameters. The respective values are shown in tables 3 a-c. The bone density, which showed normally distributed values, was in the groups with magnesium implants subjected to a decrease (table 3 a) and already differed significantly from the base value in the 4-week group in week 2 (LANd442; p = 0.002)
and week 3 (LAE442; p = 0.016). These significant changes persisted until week 4. In the 8-week groups only those animals of the LANd442-group showed significant changes in bone density from week 6 (p < 0.001), compared to the base value. In contrast, both titanium-groups showed an increase of the bone density which, however, underwent no significant changes. The groups without implants showed a contradictory behaviour. Here, in the 4-week group a tendency of the bone density to decrease was noted and to increase in the 8-week group. Both developments also produced no significant changes. In comparing the individual groups, the LANd442- group over 8 weeks already showed significant differences to the LAE442-group (p = 0.001) and the titanium-group (p = 0.003) from week 2, to the group without implants from week 6 (p = 0.029).
The values of the bone volume also followed a normal distribution and showed, in contrast to the bone density in the LAE442- and LANd442-groups, an increase (table 3 b). However, in both LANd442-groups significant differences to the base value were again observable. The 4-week group differed significantly from the base value in weeks 2 (p = 0.024) and 3 (p = 0.002), the 8-week group in week 8 (p = 0.022).
The LAE442-group over 4 weeks showed significant differences between weeks 1 and 4 (p = 0.005). Comparing the groups, significant differences were seen in week 4 between the 4-week groups of titanium and without implants (p = 0.008), and between titanium and LANd442 (p = 0.039).
The values of bone porosity were not normally distributed and, by considering the base and end values (table 3 c), underwent no or only minor changes. In the 8-week groups of LAE442, LANd442 and without implants, a tendency to increase was noted, whereas both titanium-groups as well as the LAE442-group showed a minor decrease of the porosity over 4 weeks. From these results, significant differences resulted from week 3 (p = 0.024) between the 4-week titanium group and the respective LAE442- and LANd442-groups. In the corresponding time period over 8 weeks, the titanium-group differed significantly from the LAE442-group (p = 0.014) and the LANd442-group (p = 0.024) in week 8. In the chronological sequences, no group showed significant changes.
Table 3: Development of the investigated parameters in all groups by means of three-dimensional evaluation of the in vivo µCT scans; a) bone density (in mg HA/ccm), normally distributed values; b)
bone volume (in mm³/slice), normally distributed values; c) bone porosity (in %), non-normally distributed values. Additionally stated are significances between the individual groups (a) and within a
group (b), respectively.
2.4.4 Ex vivo µ-computed tomography Bone
The results of the ex vivo µCT-investigations confirmed those of the in vivo investigations to the extent that the parameters “formation of cavities” and “periosteal
formation of new bone” occurred most strongly, whereas bone-implant contact developed only sporadically (table 4). The formation of cavities was demonstrated by all groups and the deviations between them were only slight. The contact between bone and implant only occurred distinctly in both titanium-groups (1.4 and 1.8 score points, respectively) and was rudimentary in the LANd442-group over 8 weeks (0.1 score point). Endosteal, as well as periosteal formation of new bone could be observed in all groups, except the groups without implants. For the endosteal parameter, again both titanium-groups showed the strongest formation (1.2 and 0.9 score points, respectively) and the LAE442-groups the lowest (0.3 and 0.4 score points, respectively). The results for the periosteal parameter contrasted this (both LAE442-groups: 1.6 score points; titanium: 0.4 and 0.3 score points, respectively).
Table 4: Final values of the investigated parameters in all groups by means of two-dimensional evaluation of the ex vivo µCT scans; stated are in each case (a) minimum value, (b) median and (c)
maximum value per group and parameter
group cavities bone-implant-
contact
endosteal formation of new
bone
periosteal formation of new
bone (a) (b) (c) (a) (b) (c) (a) (b) (c) (a) (b) (c) LAE442
4 weeks 1.1 1.2 2.3 0.0 0.0 0.0 0.0 0.3 0.4 0 1.6 2.0 8 weeks 0.6 1.0 1.4 0.0 0.0 0.1 0.3 0.4 0.9 0.6 1.6 2.6 LANd442
4 weeks 0.8 1.0 1.3 0.0 0.0 0.1 0.0 0.6 0.7 0.0 0.8 2.9 8 weeks 1.0 1.3 1.8 0.0 0.1 0.3 0.4 0.8 0.8 0.2 1.0 2.8 Titanium
4 weeks 1.0 1.2 2.0 0.9 1.4 2.3 1.1 1.2 1.3 0.3 0.4 0.8 8 weeks 0.2 0.8 1.3 1.0 1.9 2.7 0.8 0.9 1.2 0.0 0.3 0.7 without implant
4 weeks 0.6 0.8 1.0 - - - 0.0 0.0 0.0 0.0 0.0 0.3
8 weeks 0.4 0.7 0.9 - - - 0.0 0.0 0.2 0.0 0.0 0.0
Implant
For the degradation of the implants, a median value of 0.0 was determined in all the 4-week groups (LANd442: min 0.0, max 1.0; LAE442: min 0.0, max 0.0; titanium: min 0.0, max 0.0). In the 8-week groups, LANd442 attained a median value of 1.0 (min 0.0, max 1.0), in the LAE442- and titanium-groups the implants appeared almost
unchanged (LAE442: median 0.0, min 0.0, max 1.0; titanium: median 0.0, min 0.0, max 0.0).
2.4.5 Histological investigations Fluorescent microscopy
The highest MAR value over all time periods was achieved by the animals in the LANd442-groups, followed by the LAE442-groups (figure 4). The latter differed significantly both in the time period over 4 weeks from the titanium-group and from the group without implants (p = 0.041 and p = 0.026, respectively) as well as over 8 weeks (p = 0.009 and p = 0.033, respectively). All in all, the titanium-groups showed the lowest MAR values, apart from the time period 1 in the 4-week groups. Here the group without implants showed the lowest mineral apposition. In the 8-week group without implant, the MAR differed significantly between time periods 2 and 1 (p = 0.026).
Figure 4: Mineral apposition rate (in µm/d) per time period and experimental group; stated are in each case the mean values and significances (stars), when indicated
TRAP
The highest count of the osteoclasts and Howship’s lacunae produced over the time period of 4 weeks was attained by the LAE442-group (table 5), followed by the group
without implants, LANd442 and titanium. In contrast to this, in the time period over 8 weeks the descending order was LANd442, LAE442, titanium, without implants.
Comparing both time periods within the materials, the groups LAE442 and without implants showed higher values over 4 weeks whereas LANd442 and titanium implants activated more osteoclasts over 8 weeks (table 5). There were no significant differences between the individual time groups. However, the titanium-group and the group without implants differed significantly over 4 weeks from the respective 8-week group (p = 0.043 and p = 0.029, respectively).
Table 5: Number of osteoclasts and Howship’s lacunae per implant material and time group; stated are in each case the mean values and standard deviations, as well as significances between the time
groups of titanium (a) and the time groups without implant (b)
material 4-week group 8-week group
MV 82 55
LAE442
SD 57 39
MV 42 61
LANd442
SD 20 39
MV 11a 42a
Titanium
SD 5 24
MV 48b 12b
without implant
SD 25 5
Toluidine blue
With the help of the toluidine blue staining, it could be shown, that the cross-sections of the tibiae in all groups exhibited cavities (figure 5), whereas less cavities were formed over 4 weeks than over 8 weeks, apart from the titanium-groups. The extent of the periosteal remodelling occurred in almost all groups equally with the score value 1.0. The exceptions were both LAE442-groups and the LANd442-group over 8 weeks, which showed a stronger remodelling with 2.0 and 1.5 score points, respectively. The parameter “endosteal remodelling” was assessed in all groups with the score value 1.0. According to this, the periosteal formation of new bone occurred more strongly than the endosteal formation. In the first case, the LANd442-groups and the LAE442-group over 8 weeks demonstrated the most extensive changes, whereas in the second case, only the LANd442-group over 8 weeks attained more
than 2.0 score points. Contact between bone and implant could be observed only for both titanium-groups, however, it was strongly pronounced with 3.0 and 2.5 score points, respectively. The same holds true for the formation of a fibrous capsule, which was assessed with 2.0 score points each.
0 1 2 3 4
assesment of the bone structure
(cavities)
periosteal remodelling
endosteal remodelling
periosteal formation of new
bone
endosteal formation of new
bone
bone implant contact (trabeculae)
periimplant fibrosis
Score points
LAE442 4 weeks LAE442 8 weeks LANd442 4 weeks LANd442 8 weeks Titanium 4 weeks Titanium 8 weeks without implant 4 weeks without implant 8 weeks
Figure 5: Overview of the occurrence of the investigated parameters by means of with toluidine blue stained cross-sections of the bone.
2.5 Discussion
The aim of this study was to compare the biocompatibility of two degradable magnesium alloys with that of titanium as an established, permanent implant material. For this purpose, two materials were employed: the material LAE442, which has already been successfully tested over long periods of time (WITTE et al. 2005;
THOMANN et al. 2009), as well as the newly developed alloy LANd442.
Intramedullar implantation into a rabbit's tibia was selected as the animal model since this is established for the fundamental research of biocompatibility of various magnesium alloys (MEYER-LINDENBERG et al. 2006; THOMANN et al. 2009;
KRAUSE et al. 2010; REIFENRATH et al. 2010; HUEHNERSCHULTE et al. 2011).
Apart from implanting titanium, control tibiae were used in which the operation was
carried out but received no implant. This procedure has already been described in other studies (THOMANN et al. 2010b; HUEHNERSCHULTE et al. 2011).
By means of the clinical investigations in the current study, it was possible to establish that no differences occurred between the individual groups with regard to the redness, swelling or peripheral augmentation. Mild subcutical emphysema occurred in the 4-week LAE442-group as well as the 8-week LANd442-group in 2 and 4 tibiae, respectively. However, no occurrence was found in the tibiae of the titanium-groups or the groups without implant. Since hydrogen is formed during the degradation of magnesium (MCBRIDE 1938; WITTE et al. 2005; LI et al. 2008), it is no surprise to discover gas bubbles during the investigations of the respective alloys.
This was also described in other studies (WITTE et al. 2005; XU et al. 2007; LI et al.
2008; ZHANG et al. 2010). Similar to the above mentioned studies, the gas bubbles had absolutely no clinical effect on the current study.
In the 8-week LANd442-group, it was established that 2 rabbits each exhibited lameness lasting one day. This corresponds with a previous study of LANd442, in which the alloy was tested over a longer period of time (HAMPP et al. 2012). In that study, a single implant degraded quicker than the other implants of the same group.
It was assumed that this was the cause of the occurring lameness. Lameness also occurred using the unsuitable alloy LACer442 (REIFENRATH et al. 2010) since the implants here also degraded too quickly and thereby induced pain. In the current study, no differences could be established in the degradation behaviour of the pins, which were implanted into the debilitated animals, compared with the other implants in this group. It is possible that the formation of gas bubbles in the medullary cavity leads to changed pressure ratios and therefore to short-term soreness. It may be the case that the rabbits also received external impacts or the pain resulted from the animal's characteristic "knocking" with their hind legs in conjunction with the existing changes in the affected limbs. Nevertheless, lameness represents an undesirable effect of the implanted alloy and should therefore be negatively assessed. However, because the lameness lasts only one day, it is questionable whether this can actually be attributable to the material or whether the totality of all the circumstances caused the animals' pain.
In the current study, no greater soft-tissue reactions were established during the clinical investigations of the LAE442 alloy than those in the groups without implant and the titanium-groups. This corresponds to the previous studies of LAE442, where it showed good clinical compatibility (WITTE et al. 2007a; THOMANN et al. 2009).
For all the groups, changes could be radiologically detected. The extent of the changes is similar for all the groups, with titanium and LAE442 producing most processes within the time period of 4 weeks whereas the LANd442-groups attained the highest value over the 8 week time period. It is noticeable that both titanium- groups produced changes quite late, but then to a greater extent. Besides this, even the groups without implant also exhibited increasing curve profiles. Thus it can be assumed that the surgical method itself has a certain influence on the bone and leads to proliferating bone reactions. This agrees with an already existing study in which the influence of various medullary nailing methods were investigated in rabbits and periosteal bone regeneration was established (DANCKWARDT-LILLIESTRÖM 1969). The changes in the LANd442- and the LAE442-groups can not therefore be attributed just to the degrading implant. The growths at the implantation site constitute the largest contribution to the changes. This is less relevant to the assessment of the introduced implants' biocompatibility than to assessing the influence on the post operative healing process. The fact that the totality of changes in the 4-week groups already occurred in the first week, whereas recognisable changes in the 8-week groups were first seen in week 3 appears inexplicable. Such behaviour has not previously been described in the literature. Since it is supposed to concern the same initial material, the same behaviour in each case would be expected. A dependency of the changes on the origin or age of the animals can be excluded since no differences existed between the individual groups with respect to these factors. A varying behaviour of different material charges, which can be established using X-ray analysis, was described in a study by THOMANN (2008).
However in the current case, it concerns the same charge of each material in the corresponding time groups. It is possible that the differing behaviour can be attributed to a non-uniform composition of the implants within the charge used which can lead
to different corrosion rates (ULLMANN et al. 2011). However, this does not explain the varying behaviour of the groups without implant.
By using the cross-sectional µCT images, gas bubbles were observable over the entire test period in all animals carrying a magnesium implant. Here, variations were only very slightly pronounced in each group. Other authors concluded that hydrogen diffuses into the tissue and is therefore only visible as gas bubbles during too rapid degradation (WITTE et al. 2007c; LI et al. 2008). In the present study, the occurrence of gas bubbles is thus initially interpreted as more intense release of hydrogen with which an equilibrating ratio of regeneration to resorption of the emanating gases is associated since the total amount of gas does not increase.
Besides the formation of gas, the most pronounced change was the development of cavities which, albeit on only a small scale, also occurred for animals without implants. It can also be concluded from this that the implantation process produces changes in the bone. In contrast to this, the animals without implant exhibited no periosteal growth and only very little endosteal remodelling from week 2 to 4. On the other hand, increasing curves exist in the LANd442- and LAE442-groups for all the nominal parameters over the entire course of the investigation. This agrees with the results of the investigations using LAE442 over 6 weeks (WITTE et al. 2005) as well as the alloys LACer442 and MgCa0.8 over longer periods (REIFENRATH et al. 2010;
THOMANN et al. 2010a), which also reported periosteal bone reactions in the form of bone growth. It is concluded from this that the osseous changes in this and in the mentioned studies can be attributed to degradation induced influences of the magnesium implants, which are considered to be unavoidable. The contact between bone and implant appears to be only very slightly pronounced in the selected cross- sectional images. From week 2, both groups were assessed with an average value of 0.1 scoring points over 4 weeks. The LANd442-group developed no trabecula over 8 weeks, whereas the LAE442-group attained an average maximum value of 0.3 scoring points over 8 weeks. This is contrary to a study of LAE442 by WITTE et al.
(2005), in which bulk trabecula formation was described in guinea pigs’ femurs after 6 weeks. In the current case, it is possible that the remodelling processes in the endosteal region lead to reduced bone regeneration in the implant direction.
The relationships in the three dimensional evaluation of the in vivo µCT-scans are represented more clearly than those in the two dimensional assessment. The fall in the bone density for the LANd442- and the LAE442-groups can, on the one hand, be attributed to the formation of cavities, which were already seen in the 2D cross- sectional images. On the other hand, the drop in density could be due to regeneration of bone in the periosteal region. According to a study by FUCHS et al.
(2008), 67% of the new bone is mineralised in rabbits after 18 days. However, complete mineralisation only exists after 12 months. Thus, at the time of the current investigation, the bone could not be completely mineralised and therefore exhibits a lower density. In contrast to this, titanium induced less cavitation but significantly increased contiguous bone growth at the implant which forms a ring of bone. These bone braces are macroscopically denser than the periosteal bone tissue regenerated in, for example, LANd442 and could thereby produce an increase in the bone density.
The development of bone volume is to be seen in direct relationship to the density changes. The increasing volume in the magnesium alloy groups, as well as that in the titanium-group over 8 weeks, can also be attributed to the partially unrestricted regenerated bone tissue which, for the magnesium implants, mainly occurs periosteally and for titanium implants as peri-implant bone braces. These observations agree with a study of WITTE et al. (2005) who have described both periosteal as well as endosteal regenerated bone in magnesium alloys after 6 and 18 weeks in which the additional periosteal growth was significantly stronger. The titanium-group in the current study represents an exception to this observation over 4 weeks. This group exhibits a decrease in volume between week 3 and 4. It is possible that this observation is due to intrinsic features of the depicted µCT scan. As already described, titanium absorbs more X-rays than bone which impedes differentiating the implant from the bone (BERNHARDT et al. 2004) and possibly leads to errors in the computation. Apart from the slight rise in density already mentioned, this group shows an almost constant volume up to the 3rd week with a slightly increasing trend.
The changes in the bone porosity, which can, on the whole, be referred to as very small, can be accounted for by the factors already mentioned. This is, on the one hand, the increasing formation of cavities, which, on the other hand, is simultaneously balanced by the regenerated bone which is still cavity-free. This is most significant during observations of the titanium-group, which, as already mentioned, was only subjected to low cavitation but induced the formation of a bulk ring of bone. Unrestricted implant-bone contact is interpreted by other authors as a sign of good biocompatibility (WITTE et al. 2006). Since this regenerated bone around the implant is depicted in the µCT as very dense, thus balancing the formation of cavities, this behaviour leads on the whole to a drop in porosity.
In comparison within the two groups, the groups without implant exhibited contrary behaviour regarding the density and volume curves and showed almost no changes in the bone porosity. It can be concluded from this that certain changes within these parameters are physiological and in turn not all of the changes are, in each case, attributable to the introduced implant. However, the implants presumably reinforce the processes of bone reconstruction since these are more pronouncedly depicted for the rabbits with implants than in the groups without implant.
The ex vivo µCT-investigations confirmed the results of the in vivo investigations inasmuch that the parameters cavity formation and additional periosteal formation appeared strongly pronounced. The fact that the assessment of the bone-implant contact differs from the in vivo investigation can be explained by the higher resolution of the equipment used for the ex vivo investigations enabling a better ability both to recognise structural details and also to assess the titanium implants.
The implants were almost unchanged at the end of the testing period which was to be expected for the non-degradable titanium material. Only the LANd442-group demonstrated low levels of degradation phenomena over the 8 weeks. However, SEITZ et al. (2011) described the complete corrosion of LANd442 implants having the same geometry in one in vitro test after 18 days. Here, various studies appear to be confirmed which report that in vitro test results do not directly reflect the in vivo behaviour of materials (WITTE et al. 2006; HUANG et al. 2007; ZHANG et al. 2010).
Although WITTE et al. (2010) reported significant in vivo corrosion of cylindrical LAE442 implants after only 2 weeks.
However, Witte's implants' were, on the one hand, smaller than those used in the present study and, on the other hand, were not intramedullarily introduced into the rabbit's tibia but into the femoral condyle. This could explain the degradation behaviour deviating from the present and the other investigations (THOMANN et al.
2009; KRAUSE et al. 2010). In the current case, both magnesium alloys exhibit a promise of slow degradation which is desirable for use as osteosynthetic materials (ATRENS et al. 2011).
By means of fluorescent microscopy, it is shown that LANd442 implants induce the highest MAR at all points in time. This in turn confirms that bulk remodelling processes are active in the corresponding tibiae. LAE442 also produces a high MAR, which was significantly higher in each of the initial time intervals of the test period than those of the titanium-groups and the animals without implant. The result that LANd442 does not significantly differ from the other groups can be attributed to a higher standard deviation. Besides this, it is noticeable that, for both magnesium alloys, the MAR was larger in each of the initial time intervals than that found in the second time interval of the test period. This corresponds with the investigation of WITTE et al. (2007b), which also recorded falling MAR values due to the AZ91 magnesium alloy. In contrast to this, the titanium implants used in the current study induced a relatively low increasing MAR in the curve. The low MAR values can be explained by the bone's marked trabecula formation since little additional periosteal but much endosteal bone was developed and the MAR was determined on the periosteal bone. The groups without implant behaved differently but demonstrated almost always a higher MAR than the titanium-groups. Since the cyclic remodelling processes in the cortical bone are physiological (BALA et al. 2010), this could indicate that titanium implants diminish the bone's natural restructuring processes in favour of more marked trabecula formation. This would correspond to the VOGGENREITER’s et al. (2003) assessment that titanium is not biologically inert as has been long assumed.
On the other hand, the magnesium implants showed, on the whole, the most osteoclasts in the TRAP stained histological sections in which, as a direct comparison, more osteoclasts were counted in the group without implant over 4 weeks than in the corresponding LANd442-group. On comparing the osteoclasts' count with the density values, which were determined using the µCT computation, it was possible to establish a relationship since a more marked decrease in density accompanied a higher number of osteoclasts. The single exception to this rule was the titanium-group whose density was, as already described, subjected to an increase despite the high osteoclast activity. This can in turn be attributable to the formation of a bulk ring of bone around the implant in which no osteoclasts were found and which balances or exceeds the processes in the original bone.
The results of the evaluated toluidine blue stained bone sections also confirmed the in vivo results. Agreeing with observations from the µCT analysis, more additional bone tissue was formed periosteally than endosteally and the bone cross-sections in all the groups are pervaded by cavities. However, it was only possible to see contact between bone and implant in the titanium-groups and not in the LAE442 or the LANd442 implants, in which the latter still showed incipient trabecula formation in the µCT. Since the contact there is also only represented as minimally pronounced, it is assumed that the evaluated histological sections originate from other localisations at which no trabecula had formed. In addition to this, a moderate fibrous capsule was seen around the titanium implants by means of the histological evaluation. However, this was not seen around the magnesium implants. One such capsule, which separated the untreated titanium implant from the newly generated bone, was also described by YAN et al. (1997). Whereas VAN DER POL et al. (2010) considered the existence of fibrous tissue as unfavourable in a study of bone replacement materials, various other authors assume that a fibrous capsule around the implant will eventually be replaced by bone (YAN et al. 1997; WITTE et al. 2007c). This assumption supports the results of the current study since the formation of new bone was also only observed around the titanium implants. According to VAN DER POL et al. (2010), the fibrous capsule around the titanium would, however, argue for a poor biocompatibility of the introduced implant. In contrast to the current investigation,
magnesium implants, which are surrounded by newly generated bone, were also observed in previous studies after various time periods (WITTE et al. 2006; XU et al.
2007; ZHANG et al. 2009; ZHANG et al. 2010), sometimes even to a larger extent than a titanium implant used as a control (LI et al. 2008; CASTELLANI et al. 2011).
Although it must be taken into consideration that, in these studies, the implantation was performed in the femur and, with the exception of ZHANG et al. (2010), all the authors selected an animal model other than the rabbit. This could explain the different growth behaviour. In addition to this, all the studies mentioned above lasted for a time period of at least 9 weeks. Thus it can not be excluded that bone trabecula would also have eventually formed in the current investigation.
2.6 Conclusions
The present study showed that, in principle, both tested magnesium alloys were well tolerated. Alterations, detectable by means of imaging and histological procedures, appeared mainly in terms of periosteal formation of newly built bone. However, it could also be shown that in an identical experimental set-up, the material titanium, which is long established and in clinical use, also exerts bulk influences on the surrounding bone. Given that titanium is frequently employed as an established implant material, the potential degree of bone changes in clinical applications seems to be negligible. For this reason, an absence of effects on the bone should not also be expected from magnesium based alloys. In addition to this, the animals, which were only subjected to the surgery but received no implant in the current study, also showed active bone remodelling processes. Hence, it is assumed that by merely manipulating of the bone under surgical conditions leads to cell activation and remodelling processes and can thus not be assessed as an exclusive effect of the implant material. However, despite these observations for the two tested magnesium alloys, LAE442 seems to be the more qualified alloy since it demonstrated better clinical tolerance.
2.7 Acknowledgements
All the work for this study was carried out within the collaborative research centre 599
‘‘Sustainable bioresorbable and permanent implants of metallic and ceramic materials’’, which is funded by the German Research Foundation (DFG).
The authors would like to thank Melanie Dahms-Büttner, Melanie Kielhorn and Diana Strauch for excellent technical support.
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