und der Klinik für Hals-Nasen-Ohren-Heilkunde der Medizinischen Hochschule Hannover
Untersuchungen zur Biokompatibilität von neu entwickelten Gehörknöchelchenprothesen am
Kaninchenmodell
INAUGURAL-DISSERTATION
zur Erlangung des Grades einer Doktorin der Veterinärmedizin
(Dr. med. vet.)
durch die Tierärztliche Hochschule Hannover
Vorgelegt von Anna Christina Turck aus Mülheim an der Ruhr
Hannover 2007
Wissenschaftliche Betreuung: PD Dr. rer. nat. Karl Heinz Esser
für die Tierärztliche Hochschule Hannover
Prof. Dr. med. Thomas Lenarz
für die Medizinische Hochschule Hannover
1. Gutachter: PD Dr. Karl Heinz Esser
2. Gutachter: Prof. Dr. Andrea Meyer-Lindenberg
Tag der mündlichen Prüfung: 23.05.2007
Meinen Eltern Ulrich und Carola
Diese Dissertation basiert auf einer Veröffentlichung in einer international anerkannten Wissenschaftszeitschrift mit Gutachtersystem (peer review). Die Arbeit wurde bereits am 04.10.2006 zur Publikation angenommen.
INHALT:
Seite
I. Abkürzungen
II. Publikation
Abstract 1
Introduction 2
Materials and Methods 5
Results 8
Discussion 11
Conclusion 13
Figures 14
III. Zusammenfassung 18
IV. Erweiterte Zusammenfassung 19
V. Literatur 22
VI. Eigene Publikationen u. Koautorenschaften im Rahmen dieser Arbeit 25
VII. Danksagung 26
I. Abkürzungen
al alii
AZ Aktenzeichen
BGBL Bundesgesetzblatt
°C degree celsius
ca circa
g gram
h hour
kg kilogram
µg microgram
mg milligram
MJ megajoule
ml milliliter
mm millimeter
nm nanometer
pH pondus hydrogenii
sid semel in die
TORP total ossicular replacement prosthesis YAG Yttrium Aluminum Garnet
II. Publikation
Eingereicht in „Acta Oto-Laryngologica“; akzeptiert am 04.10.2006 Tabellen und Abbildungen befinden sich am Ende des Manuskripts
Histological Evaluation of Novel Ossicular Chain Replacement Prostheses:
An Animal Study in Rabbits
Christina Turck1, Gudrun Brandes2, Ilka Krueger3, Peter Behrens3,Hamidreza Mojallal1, Thomas Lenarz1, and Martin Stieve1
1Department of Otolaryngology, Medical University of Hannover, Germany
2Department of Cell Biology, Centre for Anatomy, Medical University of Hannover, Germany
3Institute of Inorganic Chemistry, University of Hannover, Germany
Abstract
Conclusion. The improved biocompatibility of Bioverit® II coated with a nanostructured surface shows promising qualities for use in human reconstructive middle ear surgery. In case of chitosan-hydroxyapatite composite prostheses, further investigations regarding composition of the material, degree of purity and design are necessary before clinical application.
Objective. The selection of optimal materials for reconstructive middle ear surgery continues to be a problem. In the present study novel materials are tested as total ossicular replacement prostheses (TORPs) in an animal model.
Materials and Methods. Bioverit® II coated with a nanostructured surface and chitosan- hydroxyapatite composites were placed in the middle ear of 40 rabbits. Uncoated Bioverit® II was used as control. After an implantation period of 28, 84 or 300 days petrous bones were extracted, embedded in epoxy resin and examined by light microscopy.
Results. Uncoated andcoated Bioverit® prostheses revealed a mucosal coverage and a little osseogenic response after 28 days. In case of the coated Bioverit® prostheses, the results slightly surpassed those of the plain prostheses. Chitosan-hydroxyapatite composite prostheses were mostly found dislocated after 28 days. Formations of granulation tissue and fibrotic capsules were observed around these implants. This reaction could be caused by the instability of the composite material or may be due to impurities present in the chitosan
Keywords: middle ear, TORP, biocompatibility, rabbit, nanostructure, Bioverit®, hydroxyapatite, chitosan
2
Introduction
In patients with chronic middle ear diseases such as tympanosclerosis, choleastoma and advanced otosclerosis, the ossicular chain is often destroyed. To reconstruct the sound conducting mechanism of the middle ear, a total ossicular replacement prosthesis (TORP) can be inserted between the tympanic membrane and the stapedial footplate. A variety of materials has been used for construction of TORPs. Among them titanium and bioceramics like hydroxyapatite and bioglass rate high in current use of middle ear implants [1]. Apart from basic considerations regarding biocompatibility, the reconstruction of the ossicular chain poses specific challenges, especially an optimized sound transmission, a property which relies on the mechanical and elastic characteristics of the material. As bone is a natural organic- inorganic composite structure constructed mainly from collagen and apatite, it might be hypothesized that synthetic composites could possess sound transmission properties similar to those of the bone. Whereas a secure fixation of the TORP is necessary, the ongrowth of bone could lead to the fixation of the prosthesis which would be detrimental to the efficacy for sound transmission. Therefore, the bioactivity of TORP materials has to be attenuated in comparison with bone replacement materials used in other areas of the body.
In this study, we tested three different biomaterials for TORPs in an animal study in rabbits.
Chitosan-apatite composites were chosen to mimic the composite nature of bone. As nanostructures have recently been shown to influence cell adhesion, growth and proliferation, we used standard Bioverit® glass ceramic coated with a nanoporous film as the second material. Plain (non-coated) Bioverit® prostheses were used as a reference material. Coated and non-coated Bioverit® materials had previously been studied in cell culture tests (P.
Behrens et al., unpublished data).
Glass ceramics used as biomaterials show irritation-free settling by forming a thin epithelium layer and a direct connection between bone and implant, as documented in experimental animal studies [2, 3]. There are four different types of Bioverit® glass ceramics. Bioverit® II, a machineable biocompatible glass ceramic, has already been applied successfully in middle ear surgery [4]. The implant can be adapted to different anatomical conditions with conventional instruments [5]. Bioverit® is also suitable for implantation in bacterially contaminated regions, because it inhibits the growth of gram negative bacteria [6].
In recent years, the influence of nanostructures on the adhesion, proliferation and growth of cells has been studied extensively. The term nanostructured is reserved for materials characterized by structural features on average of less than 100 nm [7]. Here, we test the influence of nanostructures in the lower nanometre range on cell viability. Compared to micro-sized ceramic formulations, nanostructured substrates showed enhanced adhesion of osteoblasts, decreased adhesion of fibroblasts and decreased adhesion of endothelial cells [8].
In addition nanostructured materials promise to avoid material instability, which is often a problem in microstructured materials [9].
Natural bone consists of organic and inorganic components, namely collagen and hydroxyapatite, respectively. Although the properties of bone rely on well-defined hierarchical and anisotropic structures, chitosan-hydroxyapatite composites can be regarded as a first approach to the organic-inorganic composite nature of bone and are being used extensively as bone substitution materials. Hydroxyapatite is a calcium phosphate ceramic with a chemical composition close to that found in cortical bone [10]. It shows the so-called osseocoalescence, which means the process of degradation, dissolution, calcification, bone- substitute and bone-ingrowth [11]. Prostheses made from pure hydroxyapatite have also gained significant acceptance in reconstructive ossicular chain tympanoplasty, as they become completely covered with epithelial cells shortly after implantation in the middle ear made [12]. Chitosan is a deacetylated derivate of chitin, commonly found in shells of marine crustaceans and cell walls of fungi. It shows various geometries and morphologies such as porous structures, suitable for cell ingrowth and osteoconduction. It has a stimulatory effect on the proliferation of macrophages and it is chemoattractant for neutrophiles. Chitosan is intrinsically antibacterial, because it disrupts the mass transport across the cell wall accelerating the death of bacteria. It was shown to promote osteoblast growth and mineral deposition in matrix in culture. There were only minimal signs of an inflammatory reaction to the material. Therefore, chitosan has a high degree of biocompatibility [13, 14], and was demonstrated as a potential material for various implant applications. The combination with hydroxyapatite has been shown to increase the osteoconductive behaviour of pure hydroxyapatite [15, 16]. The biocompatible and osteoconductive character of chitosan- hydroxyapatite composites was also demonstrated in different studies [17-21].
4
The aim of the current study was to investigate the potential of nanostructured surfaces and of the organic-inorganic composite nature of chitosan-hydroxyapatite composites as bone substitution materials in the special field of middle ear application. Here, the histological results of an animal study in rabbits are presented. Results from audiological studies will be given elsewhere.
Materials and Methods
The animal experiments (AZ 509.6-42502-04/819) were approved by the administrative district council of Hannover in accordance with paragraph 8 of the animals rights act dated on May 25, 1998 (BGBL I S.1105).
In the present study TORPs were placed in the middle ears of 40 6-month-old New Zealand White female rabbits (Charles River, Sulzfeld, Germany). Three different types of implants were used for the surgical procedures. They were constructed of chitosan-hydroxyapatite composites, Bioverit® II coated with a nanostructure and uncoated Bioverit® II for comparison (Table 1). Standard Bioverit® II TORPs were obtained from 3di GmbH, Jena, Germany. Each Bioverit® II prosthesis consists of a 2 mm long shaft, which is centrally placed on a round head of 1.7 mm diameter (Figure 1A). Nanostructured coatings on the TORPs were applied using an especially developed dip-coating procedure (P. Behrens et al., unpublished data). Bioverit prostheses were immersed in and withdrawn from an acidic water-alcohol solution containing tetraethoxysilane as a silica source and Pluronic® P123 (BASF, Ludwigshafen, Germany) as amphiphilic block copolymer. After drying, the coated prostheses were heated to 415 °C in air in order to burn off the organic amphiphile.
The nanostructure had a periodicity of ca. 5.7 nm and pores with a diameter of ca. 4 nm.
Chitosan-hydroxyapatite composite prostheses were about 1.3 mm thick cylinders and could be abridged individually (Figure 1B). These composites were obtained by first dissolving chitosan (Acros Organics, Geel, Belgium) in an acetic acid solution at pH 4; after dissolution of calcium acetate (Fluka, Switzerland) and potassium dihydrogenphosphate (Fluka), joint precipitation of chitosan and calcium phosphate was initiated by increasing the pH to 8 using potassium hydroxide solution. The resulting mass was washed with water and then mechanically densified by stuffing into a glass tube. Slow drying over two weeks at room temperature resulted in rod-like specimen as depicted in Figure 1B. The rabbits were sedated and ananaesthetized with an intramuscular injection of 1.25 mg/kg midazolam (Midazolam 5mg Curamed Injektionslösung, DeltaSelect, Pfullingen, Germany) and 25 mg/kg ketamin (Ketamin Gräub®, Albrecht, Aulendorf, Germany). To prolong anaesthetic medication 2 mg/kg propofol (Propofol-®Lipuro 1%, B. Braun, Melsungen, Germany) were applied intravenously, followed by a subcutane injection of 5 µg/kg buprenorphin (Temgesic®, Essex
6
Pharma, München, Germany). Because of the longer period for surgery, general anaesthesia was maintained by inhalation with Isofluran (Forene®, Abbott, Wiesbaden, Germany). During surgery 10 ml/kg/h electrolyte solution (Sterofundin HEG-5®, B. Braun) were infused to stabilize the rabbits` circulation systems. Then 4 mg/kg caprofen (Rimadyl®, Pfizer, Karlsruhe, Germany) was given subcutanously for three days to inhibit inflammation and pain. Metaphylaxis is given by a subcutane injection of 5 mg/kg enrofloxacin (Baytril®, Bayer Leverkusen, Germany) for ten days sid.
The tympanoplasty type III [22] was performed on the right ear so that the left middle ear of each animal remained unscathed and could serve as control. During surgery a laser microscope (OPMI Twin ER, Carl Zeiss AG, Oberkochen, Germany) was used. The external acoustic meatus was opened through a retroauricular incision. After creating a tympanomeatal flap the ossicles were detached by erbium: YAG-laser using 40 impulses at 40 MJ. The prosthesis was placed between the remaining manubrium of the malleus and the stapes footplate. In the case of the chitosan-hydroxyapatite-composite prostheses the size of each prosthesis could be adapted individually. After replacing the tympanomeatal flap siliconfoil (Bess Pro, Berlin, Germany) was placed directly to the tympanic membrane to avoid excessive formation of granulation tissue. In addition tamponades (Gelita®, Aesculap, Tuttlingen, Germany) soaked in doxycyclin (Doxycyclin-ratiopharm®SF, ratiopharm, Ulm, Germany) were deposited in the external acoustic meatus.
The prostheses remained in the middle ears for 28, 84 and 300 days (Table 1).
After sacrification with an intravenous injection of 1.6 g/kg pentobarbital (Eutha®77, Essex Pharma, Munich, Germany) petrous bones were extracted and the tympanic cavity was opened. Using a camera (Canon EOS 350 D, Tokyo, Japan), which was attached to an operative microscope (OPMI, Carl Zeiss AG) the position of the prosthesis within the bulla tympanica was documented by pictures. The specimens were fixed in 4% glutardialdehyde (Merck, Darmstadt, Germany) in phosphate-buffered saline (GIBCOTM, Invitrogen Corporation, Paisley, UK) at +4°C for 2 hours. An increasing concentration of ethanol was used to dehydrate the preparations. After drying for 8 hours at +65°C the specimens were embedded in epoxy resin (SpeciFix 20 Kit®, Struers A/S, Rodovre, Denmark) under vacuum conditions.
For histologic examination preparations were ground and polished (LaboPol-5®, Struers A/S) and then stained with Hemalum (Mayers Hämalaunlösung®, Merck, Darmstadt, Germany) and 0.1% Eosin G (Certistain®, Merck) or stained with 0.2% Eosin G and 0.6% Orange G (Certistain®, Merck) and 0.5% Toluidine Blue O (Sigma, Chemical Company, St.Louis, MO, USA). A stereoscopic microscope (Nikon® SMZ 1500, Tokyo, Japan) with 112.5 fold magnification and a light microscope (Orthoplan®, Leitz, Wetzlar, Germany) with 320 fold magnification and an external cold-light source were used to examine at least five different planes.
The development of mucosa, connective tissue, ossification and the material stability were compared and assessed gradually on a semi-quantitative scale. A digital camera (ColorviewXS, Soft Imagine Systems GmbH, Münster, Germany) was used to document the results. The images were analyzed with Analysis pro 3.2 (Soft Imagine Systems GmbH).
8
Results
All animals underwent surgery without complication. Three of 40 animals showed transient nystagmus. In all animals the incision healed by first intention and no signs of indisposition or reduction of alimentation could be observed. The macroscopic examination of the extracted temporal bones revealed that all chitosan-hydroxyapatite composite prostheses except one sample were already dislocated after 28 days (Figure 2B). During the whole implantation period dislocation did not occur in uncoated and coated Bioverit® implantations (Figure 2A) with one exception. The external auditory meatuses were found to be non irritated and free from hindering debris. The tympanic cavities generally remained free from any visible signs of infection.
Material conditions
During the implantation period the prostheses are exposed to mechanical interferences and to the defensive reaction of the body. Regarding chitosan-hydroxyapatite composite prostheses there was a marked increase of material instability. After 28 days of implantation defects and fissures were visible on microscopic examination (Figure 3B). Continuative tests pointed out that these composite seems to expand in humidity and to contract in aridity. Furthermore, small aggregates of isolated material, distributed in the tympanic cavity (Figure 3C), were detected in the tissue surrounding the chitosan-hydroxyapatite composite prostheses. This was also seen by day 84 and 300 in 4 of 6 cases of uncoated Bioverit® implants (Figure 3A), whereas in the case of coated Bioverit® implants, such aggregates were rarely visible (3 of 10 cases). No signs of erosion, abrasion or fissure formation were encountered in cases of uncoated and coated Bioverit® implants in any microscopic examination.
Mucosa
After 28 days the uncoated and coated Bioverit® prostheses displayed an extended epithelial coverage, which showed a morphology comparable to that of the adjacent mucosa lining the tympanic cavity (Figure 4). An increase in thickness of the coverage could not be found during the whole implantation period. Formation of vessels was encountered in the subepithelial connective tissue. In comparison with the uncoated implant, the nanostructured
material showed a slightly enhanced mucosal coverage, regarding the quantity of surface (Figure 4). Chitosan-hydroxyapatite composite implants showed almost no mucosal coverage during the whole implantation period. In 9 of 16 cases a fibrous capsule had developed around the prostheses before they were found to be dislocated.
Granulation tissue
There was little formation of granulation tissue in the tympanic cavity of animals that had been implanted with coated or uncoated Bioverit® after 28 days. Isolated foreign-body giant cells were mostly visible in those formations (Figure 5B). After 28 days of implantation most of the tympanic cavity was filled with a filiform network of fibrocytes (Figure 5A). During the implantation period this phenomenon decreased and fibrous connective tissue remained (Figure 5C). Isolated fibres were seen connecting the implants to the boundaries of the tympanic cavity. The main part of the fibrous connective tissue aggregated at the head of the implant. Comparing all Bioverit®-based implants after 28, 84 and 300 days connective tissue formation stagnated after 28 days of implantation.
Formation of granulation tissue occured in all cases of chitosan-hydroxyapatite composite implantation. After 28 days of implantation there was a high content of myofibroblasts (Figure 5E), which disappeared with increasing implantation time (Figure 5F). Generation of a thick fibrotic capsule (Figure 5D), which was suspended in the tympanic cavity, could already be observed after 28 days of implantation. Foreign-body giant cells did not appear.
Ossification
Plain Bioverit® implants and coated Bioverit® implants initiated a minor osseogenic response during the whole implantation period. Single regions of newly formed bone were augmented surrounding the outer surface of the head of the prostheses (Figure 6A). This effect was rarely evident on the shaft. In some cases the bone tissue was in direct contact with the implants without interposition of any other type of connective tissue (Figure 6B). Concerning the uncoated and coated Bioverit® prostheses, a slight tendency to decreased bone formation on the implants coated with a nanostructure was observed (Figure 6C). Due to the small number of animals tested, this result could not be validated statistically. There were no time-related differences in osseogenic response to plain and coated Bioverit®. With regard to the chitosan-
10
hydroxyapatite composite prostheses, already after 28 days an accretion of newly formed bone tissue was found in empty fibrotic capsules from which the prostheses had been expelled. The amount of ossification increased with time after implantation.
Discussion
Nanostructured Bioverit®
Compared with micro-sized ceramic formulations nanostructured substrates are said to enhance adhesion of osteoblasts and to decrease adhesion of fibroblasts and endothelial cells.
Primarily osteoblast adhesion occurs on particle boundaries at the surface. As nanophase materials have higher percentages of particle boundaries at the surface, this may be an explanation for the increased osteoblast adhesion measured on nanophase formulations [8].
However, the results of the present study show that both nanostructured and uncoated Bioverit® II exhibited an osseogenic response, which was only slightly minor in the case of the nanostructured prostheses. Earlier studies with Bioverit® II as partial ossicular chain replacements revealed excessive formation of new bone in guinea pigs [3], whereas other studies with rabbits showed no ossifications at all [2]. In this context, it should be noticed that human ossicles do not have any osseogenic potential post partum [23]. Therefore, in agreement with Dost et al. [3], results regarding the osseogenic response to alloplastic materials in animals should only be transferred to human applications with care. The general biocompatibility of nanophase materials is often evaluated in terms of the adherence of fibroblasts or surrounding epithelium cells. In an in vitro study on nanostructured silica coatings, produced in a different manner to those used here, cells were found to adhere only weakly to the surface and a reduction in the number of cells was encountered [24]. An in vitro test on the materials used here, employing mesenchymal progenitor cells, showed slightly reduced cell spreading, growth and proliferation on Bioverit® II coated with a nanostructure as compared to plain Bioverit® II (P. Behrens et al., unpublished data). The slight increase in mucosal coverage on coated Bioverit® prostheses, as seen in the present in vivo study, proves that biocompatibility of these modified prostheses, when measured by cell adherence and spreading, is as good as or even higher than biocompatibility of plain Bioverit®. Additionally, regarding the expanse, the coverage is already stable after 28 days. This phenomenon is similar to results of studies using currently available prostheses based on titanium [25] or hydroxyapatite [12]. However, an increase of thickness of the mucosal layer as observed in the latter studies was not revealed in the present study.
12
Chitosan-hydroxyapatite composites
By adding chitosan to hydroxyapatite the compressive modulus, yield strength and density are improved [17]. Nonetheless, some authors have ascertained that the ultimate compressive strength is lower and that the adsorption of water is increased when chitosan-hydroxyapatite materials are added to currently available bone cement [19]. The material instability and the high degree of prosthesis dislocation observed in this study could possibly be explained by the fact that these composite materials expand in humidity and contract in aridity. As surgical interventions implicate accumulation of fluids such as blood and exudate to a certain degree, there is the possibility of water adsorption, especially immediately after operation. To bypass this period until osseogenic fixation of the prosthesis, a temporary fixation should have been used. However, it remains questionable, whether fixation by malleoplatinopexy is a valid measure to avoid the dislocation of the prostheses from the stapedial footplate [26].
Chitosan is known for its high biocompatibility measured, for example, by in vitro fibroblast growth [27] or by in vivo formation of a connective tissue cover upon subcutaneous implantation [14]. In vitro studies with osteoblast cells revealed favourable cell adherence and spreading on chitosan-hydroxyapatite composites [17, 18]. The combination with hydroxyapatite has also been shown to maximize the osteoconductive properties in vivo [15, 16, 20, 21]. However, no mucosal coverage could be observed in the present study. Separated ossification centres were found surrounding the prostheses, but there was no osseous infiltration of the material in any case. This phenomenon could be explained by the dislocation of the prostheses, causing permanent mobility of the implant, which inhibits cell adhesion. VandeVord et al. observed a fibrotic capsule containing a large number of neutrophils surrounding subcutaneous chitosan implants and the deposition of collageneous material [14]. In contrast, in vivo studies with chitosan-hydroxyapatite composites have not shown any adverse effects in tissues surrounding the implants [20, 21]. The inconsistency within the reports on chitosan biocompatibility could also be caused by different qualities of this material available for purchase. As chitosan is a natural product, there is a wide range of qualities, especially regarding its purity [28]. Formation of granulation tissue and fibrotic capsules with high contents of myofibroblasts, as observed in the present study, may be caused by irritating agents present in the chitosan which was used to manufacture the prostheses.
Conclusion
In conclusion, the present histomorphological findings demonstrate that neither plain nor coated Bioverit® II gives rise to adverse effects in the tissue surrounding the middle ear implants. When compared with uncoated Bioverit® II prostheses, implants coated with a nanostructure reveal slightly enhanced mucosal coverage and, possibly, slightly reduced ossification. As this attenuated bioactivity is desirable for middle ear implants, Bioverit® II prostheses coated with a silica nanostructure appear to be a promising material for use in human reconstructive middle ear surgery.
Although evidence from the literature shows that chitosan-hydroxyapatite composites are interesting materials for a variety of implant applications, the results of this study do not favour an application of these materials in reconstructive middle ear surgery. This may be due to the transitional instability under changing conditions of humidity. However, further investigations regarding the composition of the material, the degree of purity of the chitosan used, and the design of the prosthesis are necessary before a final evaluation of this material can be given.
14
Figures
Figure 1. Middle ear prostheses.
(A) Bioverit® coated with a nanostructure; (B) chitosan-hydroxyapatite composite.
Figure 2. Position of the prostheses.
(A) correct position of coated Bioverit® prosthesis;
(B) dislocation of chitosan-hydroxyapatite composite prosthesis.
Figure 3. Material conditions.
(A) isolated material (arrows) at Bioverit® prosthesis; (B) fissure (arrow) of chitosan- hydroxyapatite composite prosthesis; (C) isolated material (arrow) at chitosan- hydroxyapatite composite prosthesis.
Figure 4. Mucosal coverage after 28 days.
(A) regular mucosa (arrow) of the middle ear. b, bone. (B) mucosal coverage (arrow) on Bioverit® coated with a nanostructure. p, prosthesis. (C) incomplete mucosal coverage on uncoated Bioverit®.
16
Figure 5. Granulation tissue.
Bioverit® prosthesis (p) coated with a nanostructure:
(A) filiform network of fibrocytes; (B) giant cells; (C) fibre rich granulation tissue (arrow) Chitosan-hydroxyapatite composite prostheses (p):
(D) fibrotic capsule; (E) cell rich granulation tissue (arrow); (F) fibre rich granulation tissue (arrow).
Figure 6. Ossification.
Bioverit® prostheses (p) coated with a nanostructure:
(A) Ossification at the head in connection with malleolus; (B) no interposition of any other type of connective tissue is observed.
Uncoated Bioverit®:
(C) slightly increased ossification (arrow).
Days of implantation:
28 84 300
Bioverit® II, uncoated 3 3 3
Bioverit® II,
nanostructured coating
5 5 5
Chitosan- hydroxyapatite composite
6 5 5
Table 1. Number of animals in each experimental group.
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III. Zusammenfassung
Christina Turck (2007): Untersuchungen zur Biokompatibilität von neu entwickelten Gehörknöchelchenprothesen am Kaninchenmodell
Hintergrund: In der rekonstruktiven Mittelohrchirurgie stellt die Wahl eines optimalen Materials für den Gehörknöchelchenersatz nach wie vor ein Problem dar. In der vorliegenden Studie wurden daher neue Materialien in Form von TORPs (total ossicular replacement prostheses) am Kaninchenmodell getestet.
Material und Methode: Bei insgesamt 40 Kaninchen wurden Prothesen aus Glaskeramik (Bioverit®), welche mit einer nanostrukturierten Oberfläche beschichtet waren, und Prothesen aus einem Chitosan-Hydroxylapatit-Komposit im Mittelohr eingesetzt. Als Kontrolle dienten dabei Prothesen aus unbeschichtetem Bioverit® II. Nach einer Implantationsdauer von 28, 84 oder 300 Tagen wurden die Felsenbeine extrahiert, in Epoxidharz eingebettet und lichtmikroskopisch untersucht.
Ergebnisse: Nach 28 Tagen zeigte sich bei den beschichteten und unbeschichteten Bioverit®- Prothesen ein Mukosaüberzug und eine leichte Ossifikationsneigung. Hierbei wiesen die nanostrukturierten Prothesen bessere Ergebnisse als die unbeschichteten auf, da die mukosaüberzogene Fläche größer und die Ossifikationsneigung geringer war. Der größte Anteil der Chitosan-Hydroxylapatit-Prothesen war bereits nach 28 Tagen disloziert. Um die Implantate hatten sich Bindegewebskapseln und Granulationsgewebe gebildet. Diese Reaktionen könnten auf die Instabilität des Materials oder mögliche Verunreinigungen zurückgeführt werden.
Schlussfolgerung: Aufgrund der verbesserten Biokompatibilität von Bioverit®-Prothesen, die mit einer nanostrukturierten Oberfläche beschichtet sind, weisen diese vielversprechende Qualitäten für den Einsatz in der humanmedizinischen Mittelohrchirurgie auf. Bezüglich des Einsatzes von Chitosan-Hydroxylapatit-Kompositen sind vorerst weitere Untersuchungen, die die Zusammensetzung, den Reinheitsgrad und die genaue Formgebung betreffen, nötig, bevor diese als Mittelohrprothesen im humanmedizinischen Bereich eingesetzt werden können.
Schlüsselwörter: Mittelohr, TORP, Biokompatibilität, Kaninchen, Nanostruktur, Bioverit®, Hydroxylapatit, Chitosan
IV. Erweiterte Zusammenfassung
Durch chronische Mittelohrentzündungen oder Otosklerose kann es zu Zerstörung oder Funktionsverlust der Gehörknöchelchenkette kommen. Um diese zu rekonstruieren, besteht daher in der Humanchirurgie weiterhin ein großer Bedarf an alloplastischen Materialien, die sowohl stabile Schallleitungseigenschaften als auch eine angepasste Bioaktivität aufweisen sollen. In der vorliegenden Studie wurden Prothesen aus Bioverit®, beschichtet mit einer nanostrukturierten Oberfläche, und Prothesen aus Chitosan-Hydroxylapatit-Komposit eingesetzt. Als Kontrolle dienten hierbei Prothesen aus unbeschichtetem Bioverit®.
Glaskeramiken, wie Bioverit® wurden bereits erfolgreich in der rekonstruktiven Mittelohrchirurgie eingesetzt [4]. In den letzten Jahren hat es vermehrt Untersuchungen zur Biokompatibilität von Nanostrukturen gegeben. Als nanostrukturiert ist dabei eine Oberflächenstruktur mit einem Größenbereich unter 100 nm definiert [7]. In diesem Größenbereich konnte ein verbessertes Zellwachstum und eine verbesserte Materialstabilität festgestellt werden [8,9].
Knochen besteht aus organischen und anorganischen Komponenten. In Nachahmung einer solchen Zusammensetzung wurden daher bei der zweiten Prothese Chitosan als organische Komponente und Hydroxylapatit als anorganische Komponente gewählt. Prothesen aus reinem Hydroxylapatit werden bereits in der Mittelohrchirurgie eingesetzt und weisen ebenfalls eine gute Biokompatibilität auf [12]. Chitosan wird aus Schalentieren gewonnen.
Die Biokompatibilität und der osteokonduktive Charakter von Chitosan-Hydroxylapatit- Kompositen wurde in Studien vielfach belegt [15-21]. Das Ziel dieser Studie war es daher, sowohl den Einfluss nanostrukturierter Oberflächen als auch das Verhalten von organisch- anorganischen Chitosan-Hydroxylapatit-Kompositen im speziellen Einsatzbereich des Mittelohres zu untersuchen.
In Rahmen der tierexperimentellen Studie erfolgte bei 40 Kaninchen im Alter von circa 6 Monaten eine einseitige Implantation. Bei den Prothesen handelt es sich um TORPs (total ossicular replacement prostheses), eine Prothesenform, bei der die gesamte Gehörknöchelchenkette ersetzt wird. Die nanostrukturierten Bioverit®-Prothesen setzen sich aus einem circa 2 mm langen massiven Schaft und einer runden Kopfplatte mit einem
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Durchmesser von circa 1,7 mm zusammen. Die nanostrukturierte Beschichtung besteht aus Siliciumdioxid, welches in einem „Dip-Coating-Verfahren“ auf die Bioverit®-Prothese aufgetragen wird. Die Chitosan-Hydroxylapatit-Prothesen bestehen aus einem ca. 1,3 mm dicken Zylinder der individuell gekürzt werden kann. Die zur Implantation erforderliche Narkose erfolgte nach Intubation mittels einer Isofluran-Inhalation. Bei der Operationstechnik handelt es sich um eine Tympanoplastik Typ 3 nach WULLSTEIN. Der Eingriff wurde ausschließlich am rechten Ohr durchgeführt. Das linke Ohr diente als Kontrolle. Die Sichtkontrolle während der Operation erfolgte durch ein Lasermikroskop. Nach Eröffnung des Gehörgangs wurden die Gehörknöchelchen mit einer Laserstärke von 40MJ bis auf die Stapesfußplatte entfernt. Die Prothese wurde mit dem Schaft auf der Stapesfußplatte und dem Kopf auf der Trommelfellebene eingestellt. Die Implantate wurden für 28 Tage, 84 Tage oder 300 Tage belassen. Nach Euthanasie der Tiere wurden die Felsenbeine entnommen und die Paukenhöhlen eröffnet. Die Präparate wurden in Glutaraldehyd fixiert, dehydriert, getrocknet und in Epoxid eingebettet. Zur histologischen Untersuchung wurden die Präparate in einem Mikroschleifverfahren geschliffen, poliert und anschließend angefärbt. Die Untersuchung erfolgte mittels Auflichtmikroskopie und externer Lichtquelle bis zu einer 320fachen Vergrößerung. Die Präparate wurden in mindestens fünf Schnittebenen durchgemustert.
Dabei wurden Mukosabildung, Bindegewebsbildung, Ossifikation, Stabilität und Oberflächenbeschaffenheit graduell bewertet und verglichen. Die Aufzeichnung und Bearbeitung der Befunde erfolgte mit einer digitalen Kamera und einem speziellen Bildverarbeitungsprogramm (Analysis pro 3.2).
Nach 28 Tagen waren bis auf eine Ausnahme alle Chitosan-Hydroxylapatit-Prothesen disloziert. Dieses Phänomen konnte bis auf eine Ausnahme weder bei den nanostrukturiert beschichteten noch bei den unbeschichteten Bioverit®-Prothesen beobachtet werden.
Zusätzlich zeigte sich bei den Chitosan-Hydroxylapatit-Prothesen eine vermehrte Materialinstabilität. Weiterführende Untersuchungen ergaben, dass dieses Komposit sich bei vermehrtem Flüssigkeitskontakt, wie er intra operationem beispielsweise durch kleinere Exsudationen gegeben ist, ausdehnt und bei Trockenheit wieder zusammenzieht. In der mikroskopischen Untersuchung der Chitosan-Hydroxylapatit-Prothesen zeigte sich eine dicke, fibröse Kapsel. Zudem kam es zu einer vermehrten Granulationsgewebsbildung in der
Paukenhöhle. Sowohl um die unbeschichteten als auch um die nanostrukturierten Prothesen hatte sich zu großen Anteilen bereits nach 28 Tagen eine mit dem Mittelohrepithel vergleichbare und somit erwünschte Mukosaschicht gebildet. Verglichen mit den unbeschichteten Prothesen zeigten die nanostrukturierten Prothesen, bezogen auf die Oberfläche, einen geringgradig vermehrten Mukosaüberzug. Am Kopf- und Fußende der nanostrukturierten und unbeschichteten Bioverit®-Prothesen kam es zu einer vermehrten Bildung von faserreichem Bindegewebe. Diese führte zu einer erhöhten Stabilität der Fixation der Prothesen. Unabhängig vom Implantationszeitraum zeigte sich in diesen Bereichen auch eine geringe Ossifikationsneigung. Eine überschießende [3] oder komplett fehlende [2]
Ossifikation konnte in der vorliegenden Studie nicht festgestellt werden. In diesem Zusammenhang sollte festgehalten werden, dass die menschlichen Ossikel post partum keine Ossifikationsfähigkeit mehr besitzen [23], was bei einer Übertragung dieser Ergebnisse auf eine humanmedizinische Applikation bedacht werden sollte.
Insgesamt hatte die Implantationsdauer keinen Einfluß auf die Epithelisierung und bindegewebige Fixation, da die oben genannten Ergebnisse bereits ab dem 28. Tag in Erscheinung traten und es zu keinen signifikanten Unterschieden zwischen den einzelnen Versuchzeiträumen kam.
Zusammenfassend lässt sich festhalten, dass sowohl unbeschichtetes als auch nanostrukturiertes Bioverit® eine gute Biokompatibilität aufweisen. Im direkten Vergleich zeigte die nanostrukturierte Prothese jedoch geringgradig bessere Ergebnisse. Bioverit®- Prothesen mit einem nanostrukturierten Siliciumdioxidüberzug sind daher als vielversprechend für den Einsatz als TORP in der humanmedizinischen Mittelohrchirurgie anzusehen.
Obwohl Chitosan-Hydroxylapatit-Komposite in der Literatur Erfolg versprechende Ergebnisse in verschiedensten Implantationsformen lieferten, sprechen die Befunde dieser Studie nicht für einen Einsatz als Gehörknöchelchenersatz. Weitere Untersuchungen bezüglich der Materialzusammensetzung, des Reinheitsgrades und der Formgebung sollten erfolgen, bevor eine abschließende Beurteilung dieses Materials zum Einsatz in der rekonstruktiven Mittelohrchirurgie durchgeführt werden kann.
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V. Literatur
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[2] Beleites E, Neupert G, Augsten G, Vogel W, Schubert H. Rasterelektronenmikroskopische Untersuchungen des Zellwachstums auf maschinell bearbeitbarer Biovitrokeramik und Glaskohlenstoff in vitro und in vivo. Laryngol Rhinol 1985; 64:217-220
[3] Dost P, Ellermann S, Mißfeldt NN, Leyen PJ, Jahnke K. Reconstruction of the stapes superstructure with a combined glass ceramic. ORL 2002;64:429-432
[4] Beleites E, Rechenbach G. Implantologie in der Kopf-Hals Chirurgie. HNO-Praxis Heute 1992;12:169-199
[5] Höland W, Vogel W, Schubert T, Schulze KJ, Carl G, Götz W, Gummel J. Structure and properties of Bioverit glass ceramics. 1990 In: Heimke G (ed) Bioceramics 2. Deutsche Keramische Gesellschaft, Cologne: 97-104
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[7] Catledge SA, Fries MD, Vohra YK, Lacefield WR, Lemons JE, Woodard S, Venugopalan R. Nanostructured ceramics for biomedical implants. J Nanosci Nanotechnol 2002;2:293-312 [8] Webster TJ, Ejiofor JU. Increased osteoblast adhesion on nanophase metals: Ti, Ti6Al4V and CoCrMo. Biomaterials 2004;25:4731-4739
[9] Trabandt N, Brandes G, Wintermantel E, Lenarz T, Stieve M. Limitations of titanium dioxide and aluminium oxide as ossicular replacement material: An evaluation of the effects of porosity on ceramic prostheses. Otol Neurotol 2004; 25:682-93
[10] Wehrs RE. Hydroxyapatite implants for otologic surgery. Otolaryngol Clin North Am 1995;28:273-86
[11] Daculsi G, Passuti P. Bioactive ceramics, fundamental properties and clinical applications: the osseo-coalescence process. In: Heimke G (ed) Bioceramics 2. Deutsche Keramische Gesellschaft, Cologne:3-10
[12] van Blitterswijk CA, Kuijpers W, Daems WT, Grote JJ. Epithelial reaction to hydroxyapatite. Acta Otolaryngol 1986;101:231-241
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[16] Muzzarelli C, Muzzarelli RA. Natural and artificial chitosan-inorganic composites. J Inorg Biochem 2002;92:89-94
[17] Zhang Y, Zhang M. Chitosan/calcium phosphate scaffolds for bone tissue engineering.
Mat Res Soc Symp Proc 2001;662:LL1.4.1-4.7.
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[19] Kim SB, Kim YJ, Yoon TL, Park SA, Cho IH, Kim EJ, Kim IA, Shin JW. The characteristics of a hydroxyapatite-chitosan-PMMA bone cement. Biomaterials 2004;25:5715-23
[20] Wang X, Ma J, Wang Y, He B. Bone repair in radii and tibias of rabbits with phosphorylated chitosan reinforced calcium phosphate cements. Biomaterials 2002;23:4167- 4176
[21] Kawakami T, Antoh M, Hasegawa H, Yamagishi T, Ito M, Eda S. Experimental study on osteoconductive properties of a chitosan-bonded hydroxyapatite self-hardening paste.
Biomaterials 1992;13:759-63
[22] Wullstein HL. 1986. Prinzipien und Definition der Tympanoplastik. In: Wullstein HL, Wullstein SA, (ed). Tympanoplastik. Stuttgart, New York: Thieme p 35-54
[23] Strohm M. 1993. Traumatologie des Ohres. In: Naumann HH, (ed). Oto-Rhino- Laryngologie in Klinik und Praxis, Bd 1: Ohr. Stuttgart: Thieme p 661
[24] Cousins BG, Doherty PJ, Williams RL, Fink J, Garvey MJ. The effect of silica nanoparticulate coatings on cellular response. J Mater Sci Mater Med 2004;15:355-9
[25] Schwager K. Epithelisierung von Titanprothesen im Mittelohr des Kaninchens.
Modellvorstellung zur Mukosaentwicklung. Laryngorhinootologie 1998;77:38-42
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[27] Ma J, Wang H, He B, Chen J. A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts. Biomaterials 2001;22:331-6
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VI. Eigene Publikationen u. Koautorenschaften im Rahmen dieser Arbeit
1. Publikationen
Mojallal H, Stieve M, Müller P, Krueger I, Witteck N, Borisov B, Turck C, Behrens P, Lenarz T. Application of Laser Doppler Vibrometry (LDV) in Middle ear Diagnostics and surgeries.
Biomedizinische Technik 50 (2005), Fachverlag Schiele und Schön
2. Poster:
Turck C, Stieve M, Brandes G, Lenarz T. Mikroschleifverfahren zur histologischen Beurteilung von neu entwickelten Gehörknöchelchenprothesen. 77. Jahresversammlung 2006 der Deutschen Gesellschaft für HNO, (24.- 28. Mai 2006), Mannheim
Stieve M, Turck C, Brandes G, Behrens P. Biomimetische Synthese von Gehörknöchelchenprothesen: Tierexperimentelle Ergebnisse von nanostrukturierten bioaktiven Materialien. 77. Jahresversammlung 2006 der Deutschen Gesellschaft für HNO, (24.- 28. Mai 2006), Mannheim
Behrens P, Müller P, Stieve M, Lenarz T, Krueger I, Mojalall H, Dimpfel F, Turck C, Witteck N, Süß B. Biomimetische Synthese von Keramiken zum Einsatz als Knochenersatzstoffe.
Innovationsforum Grenzflächenfunktionalisierung/ Biointerfaces (16.- 17. März 2006)
3. Vorträge:
Turck C, Stieve M, Brandes G, Lenarz T. Mikroschleifverfahren zur histologischen Beurteilung von neu entwickelten Gehörknöchelchenprothesen. 77. Jahresversammlung 2006 der Deutschen Gesellschaft für HNO, (24.- 28. Mai 2006), Mannheim
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VII. Danksagung
Zunächst möchte ich mich bei Herrn Prof. Dr. Thomas Lenarz und besonders bei Herrn Dr.
Martin Stieve für die Bereitstellung des interessanten Themas und die Betreuung dieser Dissertation bedanken.
Herrn PD Dr. Karl Heinz Esser danke ich für seine herzliche und unkomplizierte Betreuung und Unterstützung an der Tierärztlichen Hochschule Hannover.
Ganz herzlich möchte ich mich bei Frau Dr. Gudrun Brandes für Ihre unermüdliche Hilfe in der großen weiten Welt der Histologie und bei Herrn Peter Erfurt für seine stetige Geduld und kollegiale Zusammenarbeit im Labor bedanken.
Mein besonderer Dank gilt auch dem gesamten D1 Team des SFB 599 für die konstruktive und spannende Zusammenarbeit in diesem Projekt, insbesondere Herrn Prof. Dr. Peter Behrens für den hilfreichen Exkurs in allen Fragen der Chemie.
Danke auch dem Team des Tierlabors insbesondere Frau Kristina Krüger für die liebevolle Umsorgung meiner Kaninchen und Herrn Karl-Heinz Napierski für die kooperative und reibungslose Zusammenarbeit im Tierlabor.
Abschließend möchte ich mich bei meinen Eltern und Geschwistern und bei meinem Freund Florian Geburek ganz herzlich für Ihre reichhaltige und liebevolle Unterstützung bedanken.
