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7th Essen Symposium on Biomaterials and Biomechanics: Fundamentals and Clinical Applications of the Working Group on ”Biomaterials and Tissue Compatibility” Supported by AG Biomaterialien NRW e.V., Düsseldorf Kulturstiftung Essen e.V., Essen Life Technologies Ruhr e.V., Bochum Fahrzeugwerke Lueg AG, Center Essen

Final Program and Abstracts October, 6th – 8th, 2004 University Duisburg-Essen, Campus Essen Organizers: Alfons Fischer, Herbert Jennissen, Dieter Bingmann Scientific Board: Diethard Bergers, Mathias Epple, Heinrich Gerding (Münster), Andres Kecskemethy, Franz Löer, Horst Richter (Düsseldorf)

7th Essen Symposium on Biomaterials and Biomechanics: Fundamentals and Clinical Applications October, 6th – 8th, 2004, University Duisburg-Essen, Campus Essen

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Scope of Symposium More than 100.000 total hip or knee joints are implanted during one year in Germany. Within a time range of ten years about 5 % of those fail by aseptic loosening. The interaction of particles released from the articulating surfaces of cup and ball and the surrounding tissue is one of the major reasons. In cardiology stent related therapies have emerged into clinical application strongly since 1986. Even though many different designs, coatings, and eluted drugs have been incorporated into this treatment about 10 to 20 % of the patients suffer from restenosis. These are just two examples which depict the possibilities and limitations of modern medicine. The challenge of basic and clinical research is to understand the acting mechanisms and bring about measures to counter these problems by a well-aimed selection of suitable therapies. Due to the fact that this must incorporate specialists from all faculties this symposium has been established in order to present a forum for interdisciplinary up-to-date presentations and lively discussions. The scopes of the 7th Symposium emphasise the properties of surfaces of biomaterials and their interaction with the surrounding soft or hard tissue. This implies the characterization and the functionalisation of surfaces. For the first time special sessions on laboratory and computer simulations as well as on rapid prototyping will be provided to show the possibilities and limitations of these new methods during basic research and product development. Due to the fact that clinical experience renders the goals for any development it will by a substantial part of this symposium. The aim of all former Essen symposia has been to present the newest results as well as to initiate widespread cooperation between interested members of different faculties. Due to the special time schedule of this symposium and its poster session, which allows for stimulating and fruitful discussions, already numerous projects have been initiated. Certainly, the 7th symposium has to be seen in that course as well. Thus, we are proud and thankful that more than 50 scientists from 9 countries will present new results, ideas and concepts at our 7th symposium in Essen. Alfons Fischer


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Program Wednesday, October 6th, 2004 7:30 Registration 8:15 Welcome

Alfons Fischer (Duisburg-Essen, Germany) 8:30 - 10:00 Particles and Ions Chairpersons: Ch. Lohmann, M. von Knoch 8:30 Effects of wear particles of micro- and nanometer-scale on endothelial

cell functions in vitro Kirsten Peters (Mainz, Germany) 9:00 In-vivo micro and nano particles in blood circulation Antonietta M. Gatti (Modena, Italy) 9:30 The interaction of magnetic nanoparticles with peripheral blood cells Manuela Schwalbe (Jena, Germany) 10:00 Coffee Break 10:30 - 13:00 Particles and Ions Chairpersons: F.Löer, A.Gatti 10:30 The clinical relevance of particles and ions in orthopedics Markus Wimmer (Rush University Chicago, USA) 11:00 The morphology of wear particles in metallic biomaterials and their

direct relation to the mechanisms of detachment Robin Büscher (Duisburg-Essen, Germany) 11:30 Particle migration within rabbit-models during in vivo testing Georg Täger (Essen, Germany) 12:00 Lymphocyte-mediated Hypersensitivity in Metal/Metal Total Hip

Arthroplasty Christoph Lohmann (Hamburg, Germany) 12:30 Pharmacologic options to treat particle-induced osteolysis Marius von Knoch (Essen, Germany) 13:00 Lunch


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14:00 - 15:30 Advanced Biomaterials for Active Implants and Tissue

Engineering Chairpersons: M.Epple, M.Gelinsky 14:00 Resorbable antibiotic coatings for bone substitutes and implantable

devices Matthias Schnabelrauch (Jena, Germany) 14:30 BMPs, receptors and inhibitors. Structure and role in bone formation Walter Sebald (Würzburg, Germany) 15:00 Bone substitution materials of nanocrystalline calcium phosphate

loaded with antibiotics Thea Welzel (Duisburg-Essen, Germany) 15:30 Coffee Break 16:00 - 18:00 Advanced Biomaterials for Active Implants and Tissue

Engineering Chairpersons: W.Sebald, M.Schnabelrauch 16:00 Modified fibrin hydrogels stimulate angiogenesis in vivo: potential

application to increase perfusion of ischemic tissues Heike Hall (Zürich, Switzerland) 16:30 Alginate/calcium phosphate scaffolds with oriented, tube-like pores Michael Gelinsky (Dresden, Germany) 17:00 Implantable biomaterials made by cell growth on the collagen fibril

matrix Viorica Trandafir (Bucharest, Romania) 17:30 Silicone breast implants – Products with date of expiry! Andrea Janke (Aachen, Germany) 18:00 Poster Session and Snacks


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14:00 - 15:30 New Implants and Therapies in Opthalmology Chairpersons: H.Gerding, H.Hämmerle 14:00 Ophthalmology in Germany: cost cutting by innovative products Walter-Gerhard Wrobel (Jena, Germany) 14:30 Femtosecond laser used in ophtalmology surgery Mark Tomalla (Duisburg, Germany) 15:00 Internal tamponade of the retina Dirk-Henning Menz (Augsburg, Germany) 15:30 Coffee Break 16:00 - 17:30 New Implants and Therapies in Opthalmology Chairpersons: W.G.Wrobel, H.Richter 16:00 Bioelectrodes for chronic implants Thomas Stieglitz (St. Ingbert, Germany) 16:30 Biomaterial aspects of subretinal implants Hugo Hämmerle (Reutlingen, Germany) 17:00 Intraocular lens materials Christiane F. Kreiner (Henningsdorf, Germany) 17:30 Poster Session and Snacks


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Thursday, October 7th, 2004 8:00 - 10:00 Hard Tissue Preclinical Evaluation and Computer Simulation Chairpersons: M.Wimmer, Ch.Jäger 8:00 Development of orthopedic implants - safety concept or regulatory

hurdle Ulrich Fink (Tuttlingen, Germany) 8:30 Muscosceletal loading Georg Duda (Berlin, Germany) 9:00 Patients activities - Influence on wear behaviour of total hip

endoprostheses Michael Morlock (Hamburg, Germany) 9:30 Rapid identification of muscle activation profiles via optimisation and

smooth profile patches Andres Kecskemethy (Duisburg-Essen, Germany) 10:00 Coffee Break 10:30 - 13:00 Hard Tissue Preclinical Evaluation and Computer Simulation Chairpersons: A.Kecskemethy, U.Fink 10:30 Investigation of mechanical compatibility of implants / endoprosthesis

and bone tissue. Simulation of intelligent systems on the basis of movable cellular automaton method

Serguei Psakhie (Tomsk, Russia) 11:00 Nanocrystalline biomaterial hydroxyapatite and apatite binding in bone:

a solid state NMR perspective Christian Jäger (Berlin, Germany) 11:30 Individual biodegradable implants for cranial bone reconstruction Carsten Schiller (Duisburg-Essen, Germany) 12:00 Reconstruction of acute rotator cuff injury with new alloplastic tendon

axially fixed within a precoated bone screw experimental repair model in sheep

Martin Sellei (Aachen, Germany) 12:30 Biomaterials - Industrial quest to basic research Falko Schlottig (Oberdorf, Switzerland) 13:00 Lunch


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14:00 - 18:00 Laboratory and Computer Simulation in Soft Tissue especially

Cardiology Chairpersons: A.Kecskemethy, A.Fischer 14:00 Aspects of modelling and computer simulation of soft tissues.

Applications to arterial walls Jörg Schröder (Duisburg-Essen, Germany) 14:30 Balloon angioplasty and stenting: Mechanical and clinical aspects Gerhard A. Holzapfel (Graz, Austria) 15:00 The role of biomechanics in arterial reactions to stents James Moore (College Station, USA) 15:30 Coffee Break 16:00 - 17:30 Laboratory and Computer Simulation in Soft Tissue especially

Cardiology Chairpersons: A.Fischer, M.Epple 16:00 Various inorganic coatings for modification of the metal stents surface T. Aleksyeyeva (Kiev, Ukraine) 16:30 Thromboresistance fluorocontaining poly(urethaneurea)s: Synthesis,

properties and application O. V. Shekera (Kiev, Ukraine) 17:00 Laboratory simulation of stent fatigue Andreas Meißner (Duisburg-Essen, Germany) 17:30 Poster Session and Refreshments 19:00 Conference Dinner, City Hall Essen hosted by the mayor of the City of Essen 3rd Young Scientist Award Life Tech Ruhr Award


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14:00 - 18:00 Rapid and Virtual Prototyping in Medical Product Engineering

and Therapy Planning Chairpersons: D.Bergers, H.F. Zeilhofer 14:00 Rapid product development of a critical care board for shortening the

intensive – medical process chain Thomas Krell (Duisburg-Essen, Germany) 14:30 Reverse engineering and rapid prototyping for medical application Dieter Fichtner (Dresden, Germany) 15:00 Rapid prototyping and reverse engineering for implants Rudolf Meyer (Magdeburg, Germany) 15:30 Coffee Break 16:00 - 18:00 Rapid and Virtual Prototyping in Medical Product Engineering

and Therapy Planning Chairpersons: D.Fichtner, R. Meyer 16:00 Biocompatibility of ceramic scaffolds for bone replacement made by 3D

printing Barbara Leukers (Bonn, Germany) 16:30 Surgical navigation in space and time: what clinicians want, what is

built, what they get. Is that what they need? Wolfgang Freysinger (Innsbruck, Austria) 17:00 Computer-aided 3D-operation planning Hans-Florian Zeilhofer (Basel, Switzerland) 17:30 Biomaterials are going 3D with Layer Manufacturing Technologies (LM)

– Direct and indirect approaches Ralf Schindel (St.Gallen, Switzerland) 18:00 Poster Session and Refreshments 19:00 Conference Dinner, City Hall Essen hosted by the mayor of the City of Essen 3rd Young Scientist Award Life Tech Ruhr Award


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Friday, October 8th, 2004 8:00 - 10:00 Biomaterials, Surfaces and Cell Interaction Chairpersons: H.Jennissen, A.Curtis 8:00 3D-rapid prototyping of protein molecules relevant for biomaterials Markus Laub (Essen, Germany) 8:20 Release kinetics of BMP-2 from metal implant surfaces Maria Chatzinikolaidou (Essen, Germany) 8:40 Morphology, micromechanics and failure of bone cement Sven Henning (Halle-Wittenberg, Germany) 9:10 The osseous integration of RGD-peptide coated and non-coated metallic

implants in the adult sheep Erwin Steinhauser (München, Germany) 9:30 Measuring cell-substratum contact dynamics Mark. R. Holt (London, United Kingdom) 10:00 Coffee Break 10:30 - 13:00 Biomaterials, Surfaces and Cell Interaction Chairpersons: D.Bingmann, M. Holt 10:30 Stress sensing David Jones (Marburg, Germany) 11:00 Ionic controls of cell to cell interactions and membrane forces Adam Curtis (Glasgow, United Kingdom) 11:30 Cellular mobility on BMP covered surfaces Lars Winkler (Essen, Germany) 12:00 Toxic effects of metals Dietrich Büsselberg (Essen, Germany) 12:30 Lunch 13:30 Farewell


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Maps

Campus Essen

Rathaus City Hall

Hörsaalzentrum Lecture Halls

Mensa Cafeteria

U-Bahnhof Subway Station

Parkhaus Parking Garage

Parkplatz Parking Lot

Campus Essen


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Session: Particles and Ions Kirsten Peters (Mainz, Germany) Effects of wear particles of micro- and nanometer-scale on endothelial cell functions in vitro Antonietta M. Gatti (Modena, Italy) In-vivo micro and nano particles in blood circulation Manuela Schwalbe (Jena, Germany) The interaction of magnetic nanoparticles with peripheral blood cells Markus Wimmer (Rush University Chicago, USA) The clinical relevance of particles and ions in orthopedics Robin Büscher (Duisburg-Essen, Germany) The morphology of wear particles in metallic biomaterials and their direct relation to the mechanisms of detachment Georg Täger (Essen, Germany) Particle migration within rabbit-models during in vivo testing Christoph Lohmann (Hamburg, Germany) Lymphocyte-mediated Hypersensitivity in Metal/Metal Total Hip Arthroplasty Marius von Knoch (Essen, Germany) Pharmacologic options to treat particle-induced osteolysis


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EFFECTS OF WEAR PARTICLES OF MICRO- AND NANOMETER-SCALE ON ENDOTHELIAL CELL FUNCTIONS IN VITRO

K. Peters1, R.E. Unger1, A.M. Gatti2, E. Monari2, D.R. Haynes3, T.N. Crotti3, C.J. Kirkpatrick1

1 Institute of Pathology, Johannes Gutenberg University, Mainz, Germany 2 Laboratory of Biomaterials, Department of Surgery, University of Modena and Reggio

Emilio, Modena, Italy 3 Department of Pathology, University of Adelaide, Adelaide, Australia

The release of particulate debris by wear and corrosion of implants is an important aspect in

the pathogenesis of periprosthetic osteolysis and aseptic implant loosening [1]. In many

cases of loosened implants macrophages (Mϕ) containing these particles were detected [2].

These Mϕ appear to be crucial cell types during the observed pro-inflammatory stimulation

of the adjacent tissues [3]. Among the cellular partners of monocytes/Mϕ in inflammation

are endothelial cells (EC), which cover the inner surface of all blood vessels and are thus

present within practically every tissue. EC play a role in inflammation by expressing cell

adhesion molecules for leukocyte adherence and by the release of cytokines. Thus, EC are

important in wound healing and tissue homeostasis. However, almost nothing is known

about the effects that wear particles have on endothelial viability and function.

Recent studies indicate that, in addition to particles in the peri-implant tissues, particles (of

µm and nm size) may have been disseminated to different human tissues not only adjacent

to the implant [4]. As the source of those particles (e.g. porcelain dental bridges) and the

location of particle detection were far apart, a distribution via the blood stream is implicated.

Thus, EC which line the inner surface of blood vessels should come into direct contact with

these particles.

This in vitro study examines the effects that different particles with different sizes (of µm

and nm scale) have on the viability and function of human microvascular EC. Therefore we

studied a number of cytotoxic and pro-inflammatory aspects by different methods (on pro-

tein and mRNA-level – e.g. Elisa, cDNA array). It was shown that EC were able to internalize

large particle amounts and, depending on the particle compound, a number of particles (nm-

sized particles of Ti02, Si02, cobalt/Co) induced an early pro-inflammatory response (release

of IL-8, expression of E-selectin). Furthermore, Co-particles (nm-scale) and CoCrMo-particles

(µm-scale) induced some changes that indicated an impairment of EC viability and function.

Acknowledgements: This work was financially supported by the Deutsche Forschungsge-

meinschaft (Priority Programme Biosystem) and the European Community (QOL-2002-147)

References: [1] Zreiqat et al., Biomaterials 2003;24, [2] Willert, J Biomed Mater Res

1977;11, [3] Rogers et al., J Bone Joint Surg Br 1997;79-B, [4] Gatti & Rivasi, Biomaterials

2002;23


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MICRO AND NANOPARTICLES IN THE BLOOD CIRCULATION IN-VIVO

Antonietta M. Gatti, Stefano Montanari*, Emanuela Monari, Andrea Gambarelli

INFM, Laboratory of Biomaterials, University of Modena and Reggio Emilia, Italy * Nanodiagnostics, Italy

The study deals with a new approach to verify the presence of inorganic particulate matter in the blood circulation. By means of an innovative technique of Environmental Scanning Electron Microscopy (ESEM) and of Energy Dispersive Spectroscopy (EDS) (1,2,3), organic samples from explanted temporary vena cava filters of patients affected by blood disorders were analysed. The biological tissues adherent to the filter and the small thrombi formed in vivo were detached from the metallic structure of the device, fixed, frozen-sectioned and prepared for the histological and the electron microscopy observations. The ESEM analysis showed that both types of sample (thrombus and newly formed tissue) contained foreign, in some cases nano-sized, bodies. The chemistry of these particles was not constant, and unusual compounds containing also non biocompatible elements like Bismuth, Lead and Tungsten were detected. In a few instances, the chemistry of the particulate matter was so specific that the patient was requested if he/she had undergone a particular exposure. In all cases, the patient confirmed the hypothesis, and more than once could even indicate the exposure period.

The constant presence of those debris inside the thrombotic material we checked leads to suspect that, besides the already known factors triggering thrombosis, thrombi can originate from the interaction between the inorganic foreign bodies observed and the blood. That could account for the numerous cases of pulmonary embolism which do not show any thrombotic focus. If this hypothesis is correct, the Virchow’s Triad should be modified in order to include the foreign bodies as a fourth factor. Acknowledgements The research was supported by the European Community. Project QOL-2002-147, Nanopathology References 1. M. Ballestri, A.Baraldi, A.M. Gatti, L.Furci, A.Bagni, P.Loria, R.Rapanà, N. Carulli,

A.Albertazzi “ Liver and kidney foreign bodies granulomatosis in a patient with maloocclusion, bruxim, and worn dental prostheses” Gastroenterology , 2001, 121; 1234-38.

2. A.M. Gatti , F. Rivasi “ Biocompatibility of micro- and nanoparticles Part I in liver and kidney.” Biomaterials june 2002, vol 23

3. A.M. Gatti Biocompatibility of micro- and nanoparticles Part II in colon.” Biomaterials vol.25, 3, Feb 2004 385-392

Address: Dr. Antonietta M. Gatti INFM, Laboratory of Biomaterials Department of Neurosciences University of Modena and Reggio Emilia Via del Pozzo, 71 41100 Modena, Italy [email protected]


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THE INTERACTION OF MAGNETIC NANOPARTICLES WITH PERIPHERAL BLOOD CELLS IS

INFLUENCED BY HUMAN PLASMA IN A CELL-TYPE SPECIFIC MANNER

Manuela Schwalbe, Cornelia Jörke, Klaus Höffken, Katharina Pachmann, Joachim H. Clement

Department of Internal Medicine II (Oncology, Hematology), Friedrich Schiller University

Jena, Erlanger Allee 101, D-07740 Jena, Germany

Introduction: We could show recently, that magnetic nanoparticles interact with tumor cell

line cells and primary tumor cells in a cell-type specific manner. We studied the role of

plasma as an important fraction of human blood in order to optimize the differential labeling

of tumor cells and leukocytes derived from peripheral blood.

Methods: Leukocytes were prepared by erythrocyte lysis from whole blood samples of

healthy volunteers for in vitro experiments and from whole blood samples from patients. The

breast cancer cell line MCF-7 was kept under standard cell culture conditions. Cells were

inoculated with magnetic core/carboxymethyl-dextran nanoparticles. The average TEM-size

of the magnetite/maghemite core varyed between 3 and 15 nm. Magnetically labeled cells

were separated by MACS. The separated cells were analyzed by FACS and Laser Scanning

Cytometry (LSC). Tumor cells were detected with anti-human epithelium-antigen (HEA)-

FITC.

Results: Whole blood samples from tumor patients were separated by centrifugation. The

supernatant was defined as plasma. Then, the leukocytes were prepared by erythrocyte

lysis. The remaining cells were incubated with magnetic nanoparticles in the presence of

none, 1% or 5% plasma for 8 min and separated with our regime. The retained cells

(positive fraction) and the flow-through cells (negative-fraction) were quantified. The

leukocyte fractions showed a considerable reduction in binding of nanoparticles already after

addition of 1% plasma. In the presence of 5% plasma less than 20% of the applied cells

were separable by MACS. The amount of circulating epithelial cells within the positive and

negative fraction was estimated by LSC. In contrast to normal leukocytes the epithelial cell

fraction increased 2.5-fold in the presence of 5% plasma in comparison to the sample

without supplementation of plasma. The amount of epithelial cells enriched from patients

samples kept constant at 75% compared to the unseparated control.

Conclusion: We could show that human plasma affects the interaction of magnetic

nanoparticles with leukocytes more seriously than with epithelial tumor cell lines. This is in

concordance with previously reported results from in vitro experiments.

This work was supported by the DFG-Schwerpunktprogramm SPP1104, grant CL 202/1-1


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THE CLINICAL RELEVANVE OF PARTICLES AND IONS IN ORTHOPEDICS Joshua J. Jacobs, M.D., Markus A. Wimmer, Ph.D.

Rush University Medical Center, Chicago, IL In the vast majority of patients, permanent orthopedic implants are well tolerated, i.e. they are biocompatible. However, there is an increasing recognition that, in the long term, permanent orthopedic implants may be associated with adverse local and remote tissue responses in some individuals. These adverse effects are mediated by the degradation products of these implant materials which may be present as (1) particulate wear and corrosion debris, (2) colloidal organometallic complexes (specifically or non-specifically bound), (3) free metallic ions, (4) inorganic metal salts/oxides, and/or (5) sequestered in an organic storage form such as hemosiderin. Much of the focus on the long term biocompatibility of implant materials has centered on the metallic components because of their tendency to undergo electrochemical corrosion resulting in the formation of, at least transiently, chemically active degradation products. Concern about the release and distribution of metallic degradation products is justified by the known potential toxicities of the elements used in modern orthopedic implant alloys - titanium, aluminum, vanadium, cobalt, chromium, and nickel. Toxicity may be by virtue of (i) metabolic alterations, (ii) alterations in host/parasite interactions, (iii) immunologic interactions of metal moieties by virtue of their ability to act as haptens (specific immunological activation), anti-chemotactic agents (non-specific immunological suppression), or lymphocyte toxins, and (iv) by chemical carcinogenesis. At this time, the association of metal release from orthopedic implants with any metabolic, bacteriologic, immunologic, or carcinogenic toxicity remains conjectural since cause and effect have not been established in human subjects. This may be attributable to the difficulty of observation; most symptoms due to systemic and remote toxicity can be expected to occur in a finite frequency in any population of orthopedic patients. Particulate-induced periprosthetic bone loss leading to osteolysis and aseptic loosening – the most common complications of total joint arthroplasty – is a clear example of adverse biological effects with wear particles. The clinical significance of orthopedic wear debris accumulation at remote sites has been understood based largely on examination of lymph nodes biopsied at revision surgery or obtained for cancer-staging in patients who also happened to have a replacement arthroplasty. Numerous case reports document the presence of metallic, ceramic, and polymeric wear debris from hip and knee prostheses in regional and pelvic lymph nodes along with the findings of lymphadenopathy, gross pigmentation due to metallic debris, fibrosis, lymph node necrosis, and histiocytosis, including complete effacement of nodal architecture. Further advances in molecular biology and materials science, applied to the study of the host tissue response to implanted devices, promises to increase our understanding of the critical determinants of implant biocompatability. This will provide new opportunities for the development of improved biomaterials, novel diagnostic and screening modalities and pharmacological strategies to modify host response. Ultimately, this promises to lead to improved clinical outcomes for patients requiring implanted devices.


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The morphology of wear particles in metallic biomaterials and their direct relation

to the mechanisms of detachment

Robin Büscher, Institut für Product Engineering, Werkstofftechnik II, Universität Duisburg-

Essen

Abstract:

Wear debris is generated from implants in various physiological applications. Particularly, artificial joints (e.g. hip, knee, finger joints) and their interfaces to the human bone or the material of fixation (e.g. bone cement) undergo a dynamic wear process and therefore steadily release particles during physiological movement. Wear debris in the periarticular tissue can initiate a cellular foreign body reaction which may impair the function of the implant. Therefore, metals which are commonly used for artificial joints not only need to provide a high corrosion resistance (i.e. biocompatibility) and sufficient mechanical properties but also a high wear resistance. Nowadays, merely stainless steels and cobaltbase alloys fulfill these demands. The present study focuses on the mechanisms of wear debris generation in three metallic biomaterials under self-mating wear conditions. A conventional disc-on-pin set up was chosen to generate wear debris from a conventional medical stainless steel (X2CrNiMo18-15-3), a high nitrogen stainless steel (X13CrMnMoN18-14-3) and a wrought cobaltbase alloy (CoCr29Mo6). All tests were run for 30 h in distilled water with a Hertzian contact pressure of 370 MPa. The debris was separated from the medium by slow drying and subsequently observed by means of a SEM. Wear tracks were measured using a profilometer to obtain a wear rate. In addition, they were studied by SEM to identify the acting wear mechanisms. The worn subsurfaces were investigated with a transmission electron microscope (TEM) to gain information about the mechanisms of wear particles detachment. The results showed that the wear debris from CrNiMo steel is coarse with metallic particles in the range from 50 to 200 µm whereas the particles of CrMnMoN steel and of cobaltbase alloy are significantly finer with a size prevalence of approximately 90 nm and 100 nm, respectively. For the cobaltbase alloys, very fine particles were confirmed by the literature. However, referring to the studied stainless steels such apparent differences in particle size, shape and chemistry were not expected. TEM revealed some insights about the mechanisms of particle detachment in the austenitic metals. In a depth of approximately 30 µm, dislocation cells were observed in the CrNiMo stainless steels. Since the defect density in those cells is reportedly high, they may act as points for crack initiation. Cracks run parallel to the surface until they breakthrough to the surface to form a coarse metallic wear particle with a flaky morphology. Neither in the high nitrogen stainless steel nor in the cobaltbase alloy cell walls were formed under the wear track and hence no weak spots were generated to allow crack formation. Moreover, a dynamic recrystallization led to a nanocrystalline top surface and a gradual increase in strength towards the surface. Thereby the location of failure was moved closer to the surface leading to smaller wear particles. Due to the differences in wear mechanisms, wear rates between the tested stainless steels differed significantly and are up to one magnitude smaller for the high-nitrogen alloy. The present study showed that a change in the alloys chemical composition can achieve a dramatic reduction in wear particle size and wear rate, respectively. High-nitrogen steels generate particles which have a similar morphology and size when compared to those formed by cobaltbase alloys and hence, CrMnMoN steels may be an appropriate alternative for the use in all-metal bearings, where a low particles emission and a small particle size are crucial.


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Particle migration within rabbit-models during in vivo testing G. Taeger*, B. Hußmann*, B. Schmidt*, L. Podleska*, A. Fischer**, S. Ruchholtz*

* Klinik für Unfallchirurgie, Universitätsklinikum Essen

**Institut für Product Engineering, Universität Duisburg-Essen Introduction: Stainless steel (ASTM 316L) and titanium represent the two alloys commonly used in manufacturing of implant devices for operative fracture treatment. Reflecting that Nickel has been highly debateable for many years as to whether it can exert adverse reactions, and reflecting inferior mechanical properties of more cost-intensive titanium, laboratory research is now focusing on the development and investigation of an austenitic steel which does not contain Nickel. While biologic responses on metals from steel and titanium alloy are sufficiently characterizable in the controlled environment of in vitro laboratory research, little is known about systemic accumulation and even specific cell reactions in the clinical setting. Thus, this study aims to establish a standardized animal model with the feasibility to investigate distribution and systemic accumulation of metals and metal particles following osteosynthesis. Materials&Methods: Implant devices from stainless steel (316L), Titanium (cpTi) and nickel-free austenitic steel (CrMn) were compared in a plate osteosynthesis with bicortical screw fixation (2.7mm) following horizontal tibia-osteotomy (gap 1mm) of the right hind leg in rabbits. Groups consisted of 15 rabbits (New Zealand, 3.2kg) in the implant groups, five rabbits from other experiments on animals (no implants) served as control-group (Ctrl). Osteosyntheses were performed under intravenous anesthesia, there were no restrictions for postoperative weight bearing under systemic analgesia (Temgesic®). Animals of all groups were kept following identical conditions. Animals were sacrificed after bone healing eight weeks postoperatively. Organs (liver, spleen, kidneys, lung), pleura, peritoneum, lymph-nodes and local tissue were taken (Kera-knife®), weighed and dissolved in 65% nitric acid and 35% hydrogen (Merck p.A.). Metalspecific absorption was detected with ICP-OAS (Spectro Cirus, CCD) for titanium (Ti), chromium (Cr), nickel (Ni), manganese (Mn) and iron (Fe). Concentration of metals within tissue samples (µg/g) was calculated by converting weight of samples and dilution factor. Results are shown as mean (SEM). Results: Local tissue samples revealed highest metal concentration in the Ti-group (3900µg/g) with significant differences (p<0,001) to CrNi and CrMn (189 and 615 µg/g). Comparison of the retrieved organs showed a different degree of contamination of metal from the alloys with the spleen revealing highest levels. While Titanium was not detectable in organs of the CrNi- and CrMn-animals as expected, it was detectable only in a very limited number of Ti-animals (2/15) in a limited concentration (0.02µg/g). Concentration of Iron showed similar levels for Ti-animals compared to controls (715.1 and 740.7µg/g) but was significantly depleted in CrNi- and CrMn-animals. (426.4 and 407.4µg/g). Chromium was detectable in all animals of the CrNi- and CrMn-group (0.65 and 0.64µg/g. Surprisingly chromium was also detected in animals of the Ti-group in comparable concentrations (0.79µg/g) whereas no Chromium was detected in the controls. Similar to this finding burden of nickel was not only measured in CrNi-animals (2.1µg/g) but also in Ti- and CrMn-animals in similar concentrations (3.2 and 1.34µg/g). These values were not noticed for control animals. Discussion: The results demonstrate the feasibility of this animal model to comparatively analyze migration and distribution of metal components from different alloys within an in vivo setting on principle. It indicates an enormous local burden at the implant site following plate


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osteosynthesis with titanium. It also indicates a relevant contamination of different organs with metal components which do not seem to be part of the used implant devices but could be released from the tools used to do osteotomy and osteosynthesis. Refinement on this study will be carried out to pinpoint particle migration and specific cellular reactions in clinical orientated settings more specific in the future. NOTES:


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Lymphocyte-mediated Hypersensitivity in Metal/Metal Total Hip Arthroplasty

Ch. Lohmann


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PHARMACOLOGIC OPTIONS TO TREAT PARTICLE-INDUCED OSTEOLYSIS *von Knoch M; **von Knoch F; *Heckelei A; *Wedemeyer C; *Saxler G; ***Hilken G;

****Henschke F; *Löer F * Dpt. of Orthopaedics, University of Duisburg-Essen, Essen, Germany, **Department of

Orthopaedic Surgery, Kantonspital Chur, Switzerland, ***Central Animal Laboratory, University of Duisburg-Essen, Germany, ****Patholog. Institut am Johannisstift, Paderborn,

Germany Introduction Periprosthetic particle-induced osteolysis with subsequent aseptic loosening remains the most common and important long-term complication associated with total joint replacement. Particulate wear debris released from the implants contributes to the loosening process by activating macrophages, inducing peri-implant granulomatous inflammation which results in pronounced osteoclastogenesis and subsequent osteolysis. It is well established that the RANKL/RANK/Osteoprotegerin system plays a pivotal role in the development of osteoclastogenesis (1). RANKL binds to its membrane receptor, RANK, and promotes osteoclast activation. Osteoprotegerin (OPG) is a natural decoy receptor that competes for RANKL with RANK and thereby inhibits osteoclastogenesis and bone resorption.

Along with an increasing understanding of the molecular mechanisms behind wear debris-induced osteolysis, noninvasive treatment modalities have been evaluated for the prevention and treatment of periprothetic osteolysis. Since increased bone resorption is a key event in the process of periprosthetic osteolysis substantial research efforts have been primarily directed at blocking osteoclastic activity. Antiresorptive drugs like bisphosphonates have been shown to prevent experimental wear debris-induced bone loss, e.g. in a canine model by Shanbhag et al. (2) or in a murine model by our group (3). Based on its antiresorptive effects, OPG is another promising candidate with potential utility in the treatment of periprosthetic osteolysis. Recent experimental studies showed that OPG gene transfer protects against particle-induced osteolysis due to increased levels of endogenous OPG (4,5).

The purpose of this study is to investigated the effects of exogenous OPG

on ultra-high molecular weight polyethylene (UHMWPE) particle-induced osteolysis in a murine calvarial model. Materials and Methods Animal model: Our group recently introduced a murine calvarial model of UHMWPE particle-induced osteolysis (6) which is an extension of the well-established titanium based model (7). Our model was applied in twenty-eight C57BL/J6 mice in accordance with the institutional animal use committee approval. Animals were equally randomized to four groups. Group I underwent sham surgery only, group II received additional UHMWPE particles (about 6 x 106 particles per animal), group III and IV received particles and subcutaneous OPG (Fc-OPG, Amgen, Thousand Oaks, CA) at a daily dose of 3 mg/kg of body weight starting from day 0 (group III) or day 5 (group IV) until sacrifice. One mouse from group I did not recover from anesthesia. Retrieval and processing of specimen: Calvaria were harvested 14 days postop and prepared using techniques of undecalcified hard tissue processing. Sections were histochemically stained with Giemsa as well as for tartrate-specific acid phosphatase (TRAP). Bone histomorphometry: Bone resorption was measured as resorption within the midline suture using Giemsa staining as described previously (7). Osteoclast numbers were determined per high power field using TRAP-staining.

Statistical analysis was performed using a one-way ANOVA and a post-hoc Student’s two tailed t-test. All p-values were compared to an α-value of 0.05 to determine significance. Results Histologically, formation of fibrous and granulomatous scar tissue overlying the calvarium was found. Implantation of UHMWPE particles without any further


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intervention (group II) induced pronounced bone resorption compared to sham control levels (group I). OPG treatment starting from day 0 (group III) and delayed OPG treatment starting from day 5 (group IV) significantly diminished the particle-mediated effects. Bone resorption in midline suture was 0.094 ± 0.007 mm2 in group I, 0.25 ± 0.024 mm2 in group II, 0.093 ± 0.013 mm2 in group III and 0.093 ± 0.008 mm2 in group IV (Fig. 1). There was a statistical significant difference in midline suture bone resorption between group I and II (p=0.018), group II and III (p= 0.0015) and group II and IV (p= 0.002) (ANOVA p=0.00000004). Osteoclast numbers were 15.3 ± 3.6 in group I, 48.7 ± 7.1 in group II, 21.1 ± 5.6 in group III and 3.9 ± 10.0 in group III (Fig. 2). There was a statistical significant difference in osteoclasts numbers between group I and II (p=0.0085), group II and III (p=0.0098) and group II and IV (p= 0.0011) (ANOVA p=0.000009).

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Figure 2: Osteoclast numbers for the different groups. [mean + std err; * = p< 0.05 over group I; ** = p<0.05 over group II] Discussion Here we report that exogenous OPG treatment markedly suppressed UHMWPE particle-induced osteolysis in a murine calvarial model. According to our results these beneficial effects are primarily based on the inhibition of osteoclast formation and osteoclast function. This data provides further underlines the importance of the RANK/RANKL/OPG system during the osteoclastic cascade of particle-induced osteolysis. Of note, we found no significant differences between immediate and delayed OPG treatment regimes suggesting OPG is effective both before particle-induced osteolysis starts to occur and even after it has occurred. However, clinical data as how exogenous OPG affect wear particle-induced clinical osteolysis around total joint replacement is not available to date. Overall, OPG may be considered a candidate for noninvasive prevention and treatment of wear debris-mediated periprosthetic osteolysis after total joint replacement. References 1. Boyle W et al. Nature 423:337-42, 2003, 2. Shanbhag AS et al. Clin Orthop 344:33-43, 1997., 3. von Knoch M et al. Biomaterials 2004 in press, 4. Ulrich-Vinther et al. J Bone Joint Surg Am 84-A(8):1405-12, 2002., 5. Yang et al. Arthritis Rheum 46(9):2514-23, 2002., 6. von Knoch M et al. J Orthop Res 22:237-43, 2004., 7. Schwarz E et al. J Orthop 18:472-80, 2000. Acknowledgement: Supported by the German Research Foundation (DFG) Bonn, Germany (Grant KN 553/2).


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Session: Advanced Biomaterials for Active Implants and Tissue Engineering Matthias Schnabelrauch (Jena, Germany) Resorbable antibiotic coatings for bone substitutes and implantable devices Walter Sebald (Würzburg, Germany) BMPs, receptors and inhibitors. Structure and role in bone formation Thea Welzel (Duisburg-Essen, Germany) Bone substitution materials of nanocrystalline calcium phosphate loaded with antibiotics Heike Hall (Zürich, Switzerland) Modified fibrin hydrogels stimulate angiogenesis in vivo: potential application to increase perfusion of ischemic tissues Michael Gelinsky (Dresden, Germany) Alginate/calcium phosphate scaffolds with oriented, tube-like pores Viorica Trandafir (Bucharest, Romania) Implantable biomaterials made by cell growth on the collagen fibril matrix Andrea Janke (Aachen, Germany) Silicone breast implants – Products with date of expiry!


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RESORBABLE ANTIBIOTIC COATINGS FOR BONE SUBSTITUTES AND IMPLANTABLE DEVICES M. Schnabelrauch1, S. Vogt1, K.-D. Kühn2, U. Gopp2

1 INNOVENT Technologieentwicklung e. V., Prüssingstr. 27B, D-07745 Jena, Germany 2 Heraeus Kulzer GmbH & Co. KG, Philipp-Reis-Str. 8-13, D-61273 Wehrheim, Germany

Infections still represent a serious problem in bone surgery. Because of the poor accessibility of the infection site by systemically administered antibiotics, the local administration of antibiotics is preferred to treat or prevent bone infections. Commonly, most of the clinically used antibiotics are provided as water-soluble salts. These salts are not suitable as reservoir system and the preparation of adhering antibiotic coatings for implants or prostheses requires the use of additional components like polymers or other film-forming additives. The release of water-soluble antibiotics from inert or slowly degradable carriers is mainly controlled by diffusion processes. Therefore, it is often difficult to generate a desired release profile with regard to a special indication. Several attempts are known from the literature to overcome this problem including the use of different release-modifying polymers or polymeric top coatings and the covalent fixation of antibiotics to the substrate surface. The aim of our own work was the design of new biodegradable antibiotic coating systems in which the release profile is modulated in a simple way by complex formation of the antibiotic. For this purpose we tested the complex formation behaviour of various alkyl sulfate and fatty acid salts with gentamicin sulfate. In the case of sodium dodecyl sulfate, palmitate, and laurate, resp., sparingly water-soluble gentamicin complexes can be synthesized from aqueous solutions of the components [1, 2]. Solutions of the prepared antibiotic complexes in an appropriate solvent, e. g. in methanol, have been used for coating experiments with different substrates including calcium phosphate ceramics, bioglasses, and metals. The tested coating systems showed excellent film-forming properties without using further additives. In vitro studies on the antibiotic release from the coated devices have shown an initial burst release of gentamicin during the first day after the incubation followed by a sustained release of small gentamicin amounts up to several weeks. The amount of gentamicin released during the initial phase of the first 24 h can be properly controlled by the molar ratio of gentamicin sulfate to complex-forming agent in the coating system. The total duration of gentamicin release depends on the content of the sparingly soluble gentamicin complex in the coating. The antibiotic containing coating is completely dissolved during the release period. If necessary, the addition of further antibiotics can expand the antimicrobial effect.

Based on these findings the developed antibiotic coating systems offer a promising new approach to prophylactically reduce the incidence of bone and tissues infections as well as to treat occurring bone infections.

[1] Jurado Soler, J. Puigmarti Codina, DE 2517600 (1975).

[2] S. Vogt, K.-D. Kühn, W. Ege, K. Pawlik, M. Schnabelrauch (2003) Mat.-wiss. Werkstofftechn. 34 (2003), 1041-1047.


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BMPS, RECEPTORS AND INHIBITORS. STRUCTURE AND ROLE IN BONE FORMATION.

Sebald, W., Nickel, J., Zhang, J.-L., and Müller, Th.

Physiologische Chemie II, Theodor-Boveri-Inbstitut für Biowissenschaften (Biozentrum) der

Universität Würzburg; Würzburg 97074, Am Hubland.

Numerous BMPs and GDFs have been shown to initiate osteoinduction or osteoconduction.

Members of three subfamilies are in clinical use (BMP-2, OP-1/BMP-7) or clinical trials (GDF-

5). These protein factors have a very similar 3-dimensional structure but nevertheless they

use different receptor chains and different mechanisms of receptor activation to deliver

signals into a cell. Furthermore, structural biology and mutational/interaction analysis have

revealed specific binding modes of BMPs for type I and type II receptor chains and for

modulator proteins, as Noggin, Chordin and Gremlin, and for heparinic sites of

proteoglycans. A growing instrumentary of wild type and mutant proteins is now available to

dissect the activity of BMP proteins in tissue/cell culture and in animal disease models.

1. Scheufler, C., Sebald, W., and Hulsmeyer, M. (1999) Crystal structure of human bone

morphogenetic protein-2 at 2.7 A resolution. J Mol Biol 287, 103-115 2. Kirsch, T., Nickel, J., and Sebald, W. (2000) BMP-2 antagonists emerge from

alterations in the low-affinity binding epitope for receptor BMPR-II. EMBO J. 19, 3314-3324

3. Kirsch, T., Sebald, W., and Dreyer, M. K. (2000) Crystal structure of the BMP-2-BRIA ectodomain complex [see comments]. Nat Struct Biol 7, 492-496

4. Gilboa, L., Nohe, A., Geissendorfer, T., Sebald, W., Henis, Y. I., and Knaus, P. (2000) Bone morphogenetic protein receptor complexes on the surface of live cells: a new oligomerization mode for serine/threonine kinase receptors. Mol Biol Cell 11, 1023-1035

5. Keller, S., Nickel, J., Zhang, J. L., Sebald, W., and Mueller, T. D. (2004) Molecular recognition of BMP-2 and BMP receptor IA. Nat Struct Mol Biol 11, 481-488

6. Wurzler, K. K., Emmert, J., Eichelsbacher, F., Kubler, N. R., Sebald, W., and Reuther, J. F. (2004) Evaluation der osteoinduktiven Potenz von gentechnisch modifizierten BMP-2-Varianten. Mund Kiefer Gesichtschir 8, 83-92


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BONE SUBSTITUTION MATERIALS OF NANOCRYSTALLINE CALCIUM PHOSPHATE LOADED WITH ANTIBIOTICS

T. Welzel,1 D. Tadic,1 P. Seidel,2 E. Wüst2, E. Dingeldein,2 M. Epple*,1

1 Institute of Inorganic Chemistry, University of Duisburg-Essen, Campus Essen,

Universitaetsstrasse 5-7, D-45117 Essen, Germany 2 Coripharm GmbH & Co. KG, Lagerstrasse 11-15, D-64807 Dieburg, Germany

Mechanically stable implants for bone substitution were prepared by a continuous precipitation method that allows to prepare nanocrystalline calcium phosphate particles. These materials are close to bone mineral in composition and crystallinity and should therefore be well biodegradable in vivo, in contrast to calcined calcium phosphate ceramics (like those made from bovine bone). It is possible

1) to prepare mechanically stable objects by cold-isostatic pressing 2) to introduce porosity by mechanical drilling 3) to introduce porosity by extraction "zero-dimensional porogens" (salt crystals) and

"zero-dimensional porogens" (polymer fibres) 4) to introduce drugs (like antibiotics) into the implant.

The resulting implants could find application as drug carriers for the treatment of infections in bone. References 1. D. Tadic, F. Beckmann, K. Schwarz, M. Epple, "A novel method to produce hydroxyl-

apatite objects with interconnecting porosity that avoids sintering", Biomaterials 25 (2004) 3335-3340

2. T. Welzel, W. Meyer-Zaika, M. Epple, "Continuous preparation of monodisperse functio-nalised calcium phosphate nanoparticles with adjustable crystallinity", Chemical Communications (2004) 1204-1205.


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MODIFIED FIBRIN HYDROGELS STIMULATE ANGIOGENESIS IN VIVO: POTENTIAL APPLICATION TO INCREASE PERFUSION OF ISCHEMIC TISSUES

Heike Hall and Jeffrey A. Hubbell§ Institute for Biomedical Engineering and Department of Materials, ETH and University of Zurich, Zurich, Switzerland; § Current address: Institute for Biological Engineering and Biotechnology,

EPFL, Lausanne, Switzerland. INTRODUCTION: The lack of angiogenesis in ischemic tissues is a major health problem and many studies aim to explore strategies to locally increase blood perfusion. Our approach is to use covalently modified fibrin-based hydrogels as a matrix that induces angiogenesis in vivo. Fibrin hydrogels were covalently modified by L1Ig6, a specific receptor for αvα3-integrins (Hall et al., 2001) that is expressed on angiogenic endothelial cells (Brooks et al., 1994). In addition, L1Ig6-modified matrices were filled with growth factors VEGF-A165 or bFGF. These hydrogels were applied on growing shell-free chicken chorioallantoic membranes (CAMs) and the developing vasculature was morphometrically analyzed. Moreover, the increase in αv-integrin levels in the CAMs underlying the hydrogel implants were investigated and found to be strongly increased after CAM stimulation with L1Ig6 and/or growth factors. Modified fibrin hydrogels provide an interesting way to design an implant that can potentially be introduced at the site of ischemia, and provide a scaffold and release system for growth factors that induce specific tissue development. METHODS: CAM assay: native (Baxtor, Austria) or L1Ig6-modified matrices were filled with 0.5 µg bFGF or 0.5 µg VEGF-A165 /matrix. The matrices were applied on shell-free CAMs, and were examined after i.v. injection of fluorescein isothiocyanate dextran (SIGMA). Developing vessels were analyzed and quantified by morphometry. ELISA assay: CAMs were stimulated, extracted and homogenized (Giannopoulou et al., 2001). 100 �g total protein was substrate adsorbed, blocked in 2% BSA/TBST and Ca2+ and Mg2+. Mab αv (Calbiochem) was detected with HRP-conjugated goat anti-mouse antibodies. The data represent mean values ± SEM and were normalized to the value obtained on CAMs stimulated with native fibrin matrices. REFERENCES: Hall et al, 2001, Microvas. Res. 62, 315-326; Giannopoulou et al., 2001, Int. J. Cancer 94:690-698. Brooks et al. 1994, Cell 79:1157-114.

RESULTS

Fig 1: L1Ig6-modified fibrin matrices induce angiogenesis in vivo. Chicken chorioallantoic membranes (CAMs) were stimulated with L1Ig6-modified (a) or native fibrin hydrogels (b) and L1Ig6-modified matrices filled in addition with bFGF (c) or VEGF-A165 (d). Development of the vasculature was monitored after i.v. injection with fluorescent dextran. The scale bar represents 1 mm.

Fig 2: αv integrin is upregulated in L1Ig6 and growth factor stimulated CAMs. Chicken chorioallantoic membranes were stimulated with L1Ig6-modified or native fibrin hydrogels, both filled in addition with bFGF or VEGF-A165. The amount of αv integrin was determined on CAM extracts using ELISA. DISCUSSION: Cell-matrix interactions are responsible for the induction of angiogenesis in vivo. Increased vasculature was observed after L1Ig6 and/or growth factor stimulation and a molecular response was shown: increase in αv-integrin protein. Thusese experiments indicate that designed hydrogel implants are able to induce molecular stimuli at the site of application.

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Alginate/calcium phosphate scaffolds with oriented, tube-like pores Astrid Börner1, Gerhard Tomandl2, Wolfgang Pompe1, Michael Gelinsky1*

1 Max Bergmann Center of Biomaterials, Institut für Werkstoffwissenschaft,

Technische Universität Dresden, D-01062 Dresden, Germany 2 Institut für Keramische Werkstoffe, Technische Universität Bergakademie Freiberg,

D-09506 Freiberg/Sachsen, Germany * [email protected]

Introduction For various applications in hard tissue en-gineering porous, mineralised scaffolds are required. To enable a homogenous cell seeding, a sufficient nutrient supply during cell culture (even in big constructs) and a fast vascularisation after implantation, scaffolds with oriented and tube-like pores are necessary. New mineralised alginate scaffolds meet these demands. Materials & Methods It is known since more than 30 years that alginate forms gels with capillary-like pores when covered with solutions of di- or trivalent cations like Ca2+, Cu2+, Zn2+ or La3+ [1]. The combination of such gels with ceramic powders like hydroxyapatite (HAP), TiO2 or Al2O3, followed by sinter-ing, leads to ceramics with a regular ori-ented and channel-like pore structure which can be used as filters [2]. The com-bination of alginate and calcium phos-phates is suitable for bone tissue engi-neering, but due to the sintering the pore size gets too small for cell ingrowth and the sintered HAP is no longer degradable in vivo. Therefore we developed scaffolds with tube-like and regular pores from algi-nate/calcium phosphate composites and stabilised them by mineralisation with HAP out of solution. Results By variation of conditions of the ionotropic gelling reaction we were able to control the pore size of calcium alginate scaffolds in the range of 50-500 µm – the region which is relevant for cell seeding and vasculari-sation. An example is shown in Fig. 2 (SEM image).

Fig.: SEM image of a porous alginate/calcium phosphate scaffold (magnification 200×) Mineralisation of the porous scaffolds with hydroxyapatite out of solution led to me-chanical stabilisation in comparison to the non-mineralised samples. A further strengthening could be obtained by adding calcium phosphate particles to the alginate solution which do not affect the capillary formation. The materials showed elastic properties in the wet state and could be freeze dried for sterilisation. First cell ex-periments with osteoblasts under static and perfusion conditions occur in the moment. Because of their capillary-like pore struc-tures the new scaffolds may be a suitable material for in vitro and in vivo studies on vascularisation and angiogenesis. References [1] H. Thiele: Histolyse und Histogenese: Gewebe und ionotrope Gele − Prinzip ei-ner Strukturbildung. Akademische Ver-lagsgesellschaft, Frankfurt a. M. 1967. [2] R. Dittrich, G. Tomandl, M. Mangler: Preparation of Al2O3, TiO2 and Hydroxya-patite ceramics with pores similar to a hon-eycomb structure. Adv. Engin. Mater. 2002, 4, 487-490.

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Implantable biomaterials made by cell growth on the collagen fibril matrix

Viorica Trandafir*, Mihaela Albulescu** *INCEDTP – Division – Leather and Footwear Research Institute, Bucharest, Romania **INCDCF – Division – Chemical-Pharmaceutical Research Institute, Bucharest, Romania

This paper presents the results of some in vitro biological tests with AMATCOL collagen fibril matrix in growth of collagen-producing and hepatic cells. AMATCOL matrix was prepared by an original process, providing for a good correlation between porous microstructure, hydrating capacity, water vapour permeability, cell absorption level and matrix biodegradation rate. The resulted collagen matrix is highly compatible with collagen-producing and hepatic cells, resulting in a normal growth of them. The collagen matrix containing collagen-producing cells, grown for 7 days, was subject to some tests on animals, as a cutaneous tissue graft as compared to highly resorbed fibrous collagen dressing - PANCOL, made by ICPI and applied in treatment of skin burns. Progress in wound healing with above mentioned graft, PANCOL, and usual dressing reveals some differences, healing with cutaneous tissue graft being 15 days shorter than with PANCOL and 30 days shorter than with usual dressing. Fast skin restoration includes the histological aggregate: derma, epidermis, oil glands and hair follicles. In the near future, living tissues with human cells, characteristic of every patient and able to be grafted without any secondary effects, resulted from organogenesis tissue restoration, will be placed at the clinic disposal.


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Silicone breast implants – Products with date of expiry! Janke A1, Unbehaun N.2 Kovacs L.2,Glasmacher B.1

1Institute for Biomedical Technologies, RWTH Aachen, Germany Department for Plastic Surgery, TU Munich, Germany

Background: Breast augmentation by silicone implants get more and more popular, although complications like infections, fibrous capsulometry, silicone bleeding or even implant rupture are common. Big efforts have been made by the manufacturers to improve the situation, but despite additional coatings or modified implant surfaces and implant fillers the ratio of implant failure is still high. Lipid infiltration or silicone gel diffusion into the outer shell material are well known and susceptible of being the cause for the problems combined to silicone breast implants.

Materials and Methods:

The results reported in this study refer to the examination of 52 explanted silicone breast prostheses with an implantation time between 2 weeks and 30 years. The explants derived from different manufacturers and included textured as well as smooth surface implants. Based on a former investigation of soybean filled silicone breast implants (Klocke et al.; 5th Symposium on Biomaterials and Tissue Compatibility, Essen), mechanical properties

were determined according to ASTM 1708-79 and ASTM F 703-96. Microtensile specimens were stretched with a speed of testing of 500 mm/min at RT. To take into account a possible changing of the material properties caused by lipid infiltration or silicone gel diffusion into the outer shell material, samples were additionally pre-treated by soxhlet-extraction before the tensile test. The results were compared to the non pre-treated samples.

Results:

Main reason for the explantation of the tested silicone implants was the development of a painful fibrous capsule. Almost 8 % showed silicone bleeding or even rupture of the implant shell. So far, there is no clear correlation between material degradation and increasing implantation time. Tensile strength and elongation at break tend to result in a decrease with increasing implantion time for the textured implants, whereas smooth implants show a more indefinite behavior. Pre-treatment of the samples before the tensile testing results generally in higher outcomes for tensile strength and elongation at break independent of the kind of implant surface. Furthermore the weight loss caused by the extraction is with 17.7% considerably higher with smooth surface implants as compared to 10,7% of textured surface implants.

Conclusion:

The investigation will be extended to suitable reference materials and a larger number of prostheses of a single manufacturer. Clinical data and composition of extracts are evaluated in ongoing tests.


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Session: New Implants and Therapies in Opthalmology Walter-Gerhard Wrobel (Jena, Germany) Ophthalmology in Germany: cost cutting by innovative products Mark Tomalla (Duisburg, Germany) Femtosecond laser used in ophtalmology surgery Dirk-Henning Menz (Augsburg, Germany) Internal tamponade of the retina Thomas Stieglitz (St. Ingbert, Germany) Bioelectrodes for chronic implants Hugo Hämmerle (Reutlingen, Germany) Biomaterial aspects of subretinal implants Christiane F. Kreiner (Henningsdorf, Germany) Intraocular lens materials


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Ophthalmology in Germany: cost cutting by innovative products

Dr.Walter-G. Wrobel Chief Technology Officer

Carl Zeiss Meditec AG Jena

Rising health care costs in many countries put a pressure on the health care budget. The health care industry is probably the biggest industriy in many developed countries. In the U.S., 14% of GNP is spent for health care. Due to demographics, the health market is expected to grow for another 100 years. Ophthalmology is a small part of this industry (in the %-range), but nevertheless an important sector, since blindness leads to a severe loss in quality of life as well as drastic economic consequences (loss of income, need for support personnel). In the last decades, Ophthalmology has increased its scope by making new treatments available for hitherto uncurable diseases. At the same time, the efficiency of existing diagnostic and therapeutic procedures has increased remarkably. We will discuss examples for different approaches to increase efficiency in Ophthalmology. Expenses can be reduced by making devices more compact, reduce power consumption and thereby increase reliability (Example: laser photocoagulators). Efficiency can be increased by saving precious doctor’s time. Procedures can be simplified, streamlined, making them less complex and easier to handle. In this way, procedures can also be delegated to less-educated (=cheaper) assisting personnel. An successful approach is the work station concept, where several diagnostic procedures for one disease are combined in one device (Example: IOLMaster, CRSMaster, Digital Slitlamp). Efficiency can also be increased by developing treatment methods which are more automated, and less dependent on the skills of the surgeon. (Example: LASIK with FS-Lasers). One of the main efficiency drivers could be SW and telemedicine. Compared to other industries, the SW and IT expenses in Health are below average. SW and telemedicine can reduce double work, wrong diagnosis, and paper volume. (Example: Fundus imaging). More efficiency means not only cost reduction, but primarily better care and more time the ophthalmologist can spend with the patient – his main objective. However, especially in Germany, state regulations, ethical guidelines and capping of health care costs create a situation which is blocking the road to more efficiency. This does not allow cost containment, but also could limit the quality of health care.


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Femtosecond laser used in ophthalmology surgery

Mark Tomalla M.D.

Ev.u. Joh. Klinikum Duisburg Nord, Klinik für Refraktive Chirurgie, Fahrnerstr. 133, 47169 Duisburg, Germany

Purpose: We started with the 20/10 PerfectVision femtosecond laser in June 2004. We compared laser in situ keratomileusis (Lasik) results obtained with the femtosecond laser and the mechanical microkeratom (Hansatom, Bausch & Lomb, Inc.). Setting: Ev. Klinikum Duisburg, Clinic for refractive surgery, Fahrnerstr. 133, 47169 Duisburg Methods: The analysis compares Lasik outcomes with the 20/10 Perfect Vision femtosecond laser and with the Hansatom mechanical microkeratom. The two groups were matched for the enrollment criteria and were operated under similar conditions by the same surgeon. Results: There were 20 eyes in the femtosecond laser group and 20 eyes in the mechanical microkeratom group. One day postoperatively the visual acuity in both groups were similar to each other. According to the results of two months we found a significant difference in astigmatic between both groups. The femtosecond laser showed a better astigmatic neutrality. We also have a thinner flap and no epithelium growth into the interface. Conclusion: The 20/10 Perfect Vision femtosecond laser demonstrates much more predictable flap thickness, a higher astigmatic neutrality and decreases epithelial injury in comparison to the mechanical microkeratom.


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Internal tamponade of the Retina

Dirk-Henning Menz (1), Joachim H. Dresp (2) (1) Pharmpur GmbH, Holzweg 25 – 27, 86154 Augsburg, Germany,

e-mail: [email protected] (2) Bausch & Lomb GmbH, Hans-Riedl-Str. 7-9, 85622 Feldkirchen, Germany

Recently the portfolio of ocular tamponades for internal treatment of retinal detachment was extended by two new groups of substances. In addition to the well known ocular endotamponades like fluorinated gases, perfluorocarbon liquids and silicone oil, partially fluorinated liquids and the so called „heaver than water“ silicone oils – mixtures of silicone oil and partial fluorinated liquids – have been introduced into vitreoretinal surgery and are available now as CE-marked products. The peculiarity of liquid ocular endotamponades is, that they are used as intraoperative instruments, using their liquidity on the one hand for an easy application and an extensive flattening of the retina. On the other hand the stability of the shape of the bubble predetermines these fluids as endotamponades. Basic prerequisites are a high chemical inertness and an excellent purity over the whole time of application, combined with a high interfacial tension against water. Differences in their behaviour as well as in their reaction can be traced back to their chemical structure. The differences in specific gravity in combination with different surface/interfacial tensions result in different bubble shapes and are used in vitreoretinal surgery. In combination with other features like viscosity they are also responsible for special side effects (tank lorry effect). With the increasing number of ocular endotamponades and their fine-tuned features new applications will be possible. Additionally effects of interactions, neglected in the past, should now be considered. The improvements in the last decade in manufacturing of ocular endotamponades ensure the high quality of the commercially available CE-marked products. However, the problem with impurities still remains but nowadays it exists in the form of contaminations by materials and instruments used during the surgery, which can create problems, especially in the case of a long term tamponade. It has to be considered that with an increasing tamponade time the critical concentration level of impurities drops down to a ppm level. The evaluations of analytical tests of explanted silicone oil samples demonstrate the relations between the concentration of impurities and side effects like emulsification and stickiness. These test results show also the important role of individual conditions of the patient. Therefore especially a decrease in the surface tension of the water phase inside the vitreous cavity and the alteration of surface behaviour of the tamponade media caused by the interaction with other materials used intraoperatively is extremely important, especially during a direct exchange. In order to understand the tamponade effect as well as the most important side effects like emulsification of silicone oil or the adherence during removal, the interaction of all ocular endotamponade media, with instruments and equipment used during surgery, and with the tissue (retina, ILM) must be taken into consideration. First approaches to simulate these processes using additional surface related parameters will be presented and discussed and some aspects of that will be demonstrated by high-speed video sequences of in vitro experiments.


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Bioelectrodes for Chronic Implants Thomas Stieglitz1

Neural Prosthetics Group, Fraunhofer Institute for Biomedical Engineering

Ensheimer Str. 48, 66386 St. Ingbert, Germany Phone: + 49 6894 980 160; Fax: + 49 6894 980 400;

email: [email protected] Electrodes are the key components in electrically active implants to interface the technical system with the biological tissue. While the recording of bioelectrical signals can be predominantly described as information transfer, electrical stimulation needs detailed consideration of the charge transfer processes of the phase boundary. Chronic implants have to be designed to be functional and safe over decades which is commonly summarized in the term “biocompatibility”. The biocompatibility of an electrical active chronic implant does not only include the non-toxicity of the materials that are in direct contact with the surrounding tissue, i.e. surface biocompatibility but also the mechanical interaction between implant and target tissue due to its geometry and flexibility which is commonly known as structural biocompatibility. The biostability of insulation and electrode materials is mandatory to guarantee the functionality of the implant system. Most implants fulfill these target specifications by using titanium or ceramic housings to protect the implant electronics against moisture and ions and metal sheets as electrodes for safe charge transfer over the life-time of the implant. Latest developments of advanced implants make use of micromachining technologies to cope with anatomical constraints and physiological requirements. The approach of an epiretinal vision prosthesis as a prominent example for an advanced implant shall illustrate the possibilities and challenges of miniaturized stimulation electrodes integrated into a biomedical microsystem. We chose an approach that used polyimide as substrate and insulation material. Electrodes were monolithically integrated on flexible substrates by means of thin-film technology. The substrates served as carrier for the hybrid assembly of electronic components and integrated circuits of the implant electronics. The geometry of the electrode carriers was designed to match the shape of the eye globe. So far, 24 electrodes have been integrated into an array of 4 mm diameter. The basic version of the electrodes consisted of a platinum thin-film metallization of 70 µm diameter and 300 nm thickness. Coatings with platinum black or iridium oxide were deposited optionally. In vitro investigations as well as acute and chronic implantations were performed to evaluate the biocompatibility and functionality of the electrodes. Cytotoxicity tests according to ISO 10993 proved that neither the material nor the process technology led to toxic reactions in contact with mouse fibroblasts (L 929) and oligodendroglia cells (OLN 93). Supramaximal excitation of retinal ganglion cells was obtained with stimulation pulses below 100 µA at pulse widths of 250 µs. So far, the chronic behavior of implants was only tested with electrically inactive implants. Electrophysiological investigations with flash light as well as histological evaluation of the retinae six months after implantation showed no significant alterations in the physiology and anatomy. The presented concept of a flexible biomedical microimplant proved its potential for chronic use in advanced implants. Acknowledgement: This work was supported by the German Ministry of Education and Research (BMBF) within the Retina Implant Project (01 IN 501 Q, 01 KP 0003). 1 after October 1st, the author will be with the Chair of Biomedical Microtechnology, Institute for Microsystem Technology (IMTEK), University of Freiburg, Georges-Koehler-Allee 102, 79110 Freiburg, phone: +49 761 203 7471; email: [email protected]


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Biomaterial aspects of subretinal implants

Hugo Hämmerle1, Karin Kobuch3, Konrad Kohler2, Wilfried Nisch1, Helmut Sachs3, Martin Stelzle1

1 Natural and Medical Sciences Institute, Markwiesenstraße 55, D-72770 Reutlingen, Germany, Fax.: 0049 (0) 7121 51530 16, e-mail: [email protected]

2 University Eye Hospital, Schleichstraße 12-16, 72076 Tübingen, Germany 3 University Eye Hospital, Regensburg, Franz-Joseph Strauß Allee 11, 93042 Regensburg,

Germany Abstract

Micro-photodiode arrays as well as advanced hybrid devices comprising active stimulation chips have been developed to replace degenerated photoreceptor cells in the retina. In order to deliver stimulation current to the adjacent tissue via micro-electrodes, a multitude of micro-feedthroughs has to be integrated into the encapsulation. Long-term biostability versus corrosion in vivo is a fundamental and demanding requirement of such an implantable device. Biostability of micro-photodiode arrays (MPDA) was investigated in vitro and in vivo. No significant damage was found on chips immersed for up to 21 months in saline solution. In vivo, however, the silicon oxide passivation layer of the chip was dissolved within a period of about 6 to 12 months. Subsequently, the underlying silicon was corroded as well. In contrast, stimulation electrodes consisting of titanium nitride were well preserved both in vitro and in vivo. The deterioration of the electrical properties of the micro-photodiodes correlated with the morphological damage observed. Strategies aiming at the development of an improved biostable encapsulation of neurotechnological implants have been investigated and an improved encapsulation technology has been successfully established.

Fig.1: Corrosion observed on microphotodiode arrays after sub-retinal implantation for 0.5 up to 28 months. Encapsulation consisted of low-temperature deposited silicon oxide (TEOS).

Fig.2: Advanced encapsulation after 12 months in vivo: neither passivation layer (left, light microscopy) nor micro-electrodes (right, SEM micrographs) show any deterioration in contrast to conventional passivation materials.

Light microscopy

SEM

200µm


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Intraocular lens materials

Dr. C. F. Kreiner Acri.Tec GmbH

Neeuendorfstrasse 20a D-16761 Hennigsdorf

Introduction During the last 10 years ophthalmosurgical technology improved significantly. New techniques required advanced materials suitable for intraocular implants. Especially foldable polymers were required to enable the surgeon to carry on a small incision cataract surgery. Many different intraocular lenses are offered, the majority of which seem to be modifications of the same basic copolymer. To understand the suitability and evaluate the clinical relevance of the different intraocular lenses the specific physio-chemical properties have to be understood.

Materials and Method

Actually three different copolymers are clinically used: Polymethylmethacrylate (PMMA), foldable Acrylate and Silicone elastomers.

The physical-chemical properties as well as the biocompatibility of these basic materials and their modifications are characterized and differences identified.

Results

Despite the fact that many trade marks exist in the field of intraocular lenses, the materials of these products can be traced back to only few basic copolymers.

Discussion

The development of new ocular implant materials is limited by physiological and clinical requirements which have to be met. Therefore, even materials very recently introduced can be traced back to well known basic polymers.


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Session: Hard Tissue Preclinical Evaluation and Computer Simulation Ulrich Fink (Tuttlingen, Germany) Development of orthopedic implants - safety concept or regulatory hurdle Georg Duda (Berlin, Germany) Muscosceletal loading Michael Morlock (Hamburg, Germany) Patients activities - Influence on wear behaviour of total hip endoprostheses Andres Kecskemethy (Duisburg-Essen, Germany) Rapid identification of muscle activation profiles via optimisation and smooth profile patches Serguei Psakhie (Tomsk, Russia) Investigation of mechanical compatibility of implants / endoprosthesis and bone tissue. Simulation of intelligent systems on the basis of movable cellular automaton method Christian Jäger (Berlin, Germany) Nanocrystalline biomaterial hydroxyapatite and apatite binding in bone: a solid state NMR perspective Carsten Schiller (Duisburg-Essen, Germany) Individual biodegradable implants for cranial bone reconstruction Martin Sellei (Aachen, Germany) Reconstruction of acute rotator cuff injury with new alloplastic tendon axially fixed within a precoated bone screw experimental repair model in sheep Falko Schlottig (Oberdorf, Switzerland) Biomaterials - Industrial quest to basic research


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DEVELOPMENT OF ORTHOPEDIC IMPLANTS – SAFTEY CONCEPT OR REGULATORY HURDLE

Ulrich Fink; R&D, Aesculap AG & Co. KG, Tuttlingen, Germany

More recently various serious systemic failures with orthopedic implants have been reported in the market place. We have seen for example toxicological surface contamination on porous coated acetabular components (FDA recall No. Z-404-1 ff. dated 18th July 2001) and wear problems with highly crystalline phase modified polyethylene (Scott et al, 2000). In addition we have seen corrosion induced loosening of cemented titanium stems in hip arthroplasty (Willert et al, 1996) and we saw mechanical brake down of a PMMA bone cement due to problems with the glass transition temperature (Abdel-Kader et al, 2001). All the above mentioned products failed despite intensive laboratory testing and the approval of regulatory or notified bodies either in Europe or in the United States. The result on the one hand was harm to the patient, but also on the other hand it affected the medical device company due to recourse claims from the patients and in the midterm, the loss of confidence from the surgeons. So as to receive the authorization to sell from the certifying or notified bodies, the medical device company has to document the safety and efficiency of the product as well as the reproducibility in production. For all these aspectsit is vital to perform a risk analysis so as to get an idea about the worst case scenario. In addition several organizations for the standardization of test procedures and design features do exist. “Specialists” are consulted by these organizations in order to define the criteria for testing. Nevertheless the aforementioned systemic failures still occurred. In contrast there are several restrictions for products from the government or notified bodies which the producers themselves don’t really understand. Examples of such restrictions are those imposed on pedicle screws and on cementless joint replacements. A fair but critical evaluation for a new product can only really be made for variations on existing products. The key features require for registration is the risk analysis and a knowledge and a knowledge of the relevant failures of medical devices in the past. CONCLUSION Every clinical innovation has to be proven by actual clinical data. To ensure risk minimization pre-clinical laboratory testing is an absolute prerequisite for this comparison. The comparison, however cannot only be made based on such laboratory tests since history has shown that many uncertainties in the risk analysis do exist. Computer models can presently only provide a hint towards the approval of a product and must in any case be validated by mechanical and/or in-vitro testing. In order to limit the effort and expenses in terms of the regulatory work, and, more importantly, to provide the highest possible security for the patient, a well defined clinical procedure for the assessment of reproducible clinical outcomes for standard indications has to be defined. To judge these comparative results it is necessary to develop improved measurement techniques for clinical outcomes. REFERENCES Abdel-Kader, K.F. et al (2001). J. Arthroplasty, 16, 811-819 Scott, D.L. et al (2000). J. Arthroplasty, 15, 35-46 Willert, H.G. et al (1996). Clin. Orthop., 333, 51-75


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Musculoskeletal Loading Georg Duda

Charité, University Medicine Berlin, Free and Humboldt-University of Berlin, Augustenburger Platz 1, 13353 Berlin, Germany

In biomechanics, a great deal of research has been conducted in an effort to characterize the mechanics and interaction of the soft and hard tissue structures. Unfortunately, many investigators tend to treat the muscles and bones as static, separate and independent structures. However, in order to fully understand how loads are transmitted within the body the mechanical interaction of these structures as well as the biological consequences of that interaction must be considered. Surgical intervention of a joint routinely endeavours to recreate a physiologically normal joint loading environment. The loading conditions resulting from osteotomies, fracture treatment and arthroplasties are considered to have an impact on the long term clinical outcome of a joint. Using a previously validated musculoskeletal model of the human lower limb, our studies have focused on the tibio-femoral and hip joint forces that occur during the common daily activities of walking and stair-climbing.

The average resultant peak tibio-femoral contact force during walking was found to average 3.2 times body weight (BW) across a group of patients. Knee contact forces were considerably larger than hip contact forces. Inter-individual variation proved larger than the variation of forces for each patient repeating the same task. Forces through the joint were considerably larger during stair climbing than during walking: the average maximum knee force during stair climbing was 5.4 BW although peaks of up to 6.2 BW were calculated for one particular patient. Average anterior-posterior peak shear forces at the knee of 0.6 BW were determined during walking and 1.3 BW during stair climbing. These results demonstrate the importance of muscular activity in adding to body weight to create high forces across a joint. Since musculoskeletal loading directly influences bone healing, internal loads in patients are important measures in therapy planning and patient treatment. Interfragmentary movements and ground reaction forces were measured in patients with tibial osteotomies stabilized by Ilizarov-hybrid constructs until either bone union or conversion to internal fixation occurred. In all patients, muscle co-contraction led to interfragmentary movements comparable to those during level walking. Although patients showed differences in healing characteristics due to their initial injury and treatment, all patients showed in vivo challenging mechanical conditions.

Our studies illustrate the close interaction of bones and muscles in terms of the mechanical conditions of the musculoskeletal system. For the lasting function of an artificial joint or for the healing of fractured bones the mechanical loading of the biological tissue are essential. In that sense, healing of the soft tissue structures such as muscles is mandatory to re-establish moderate mechanical loading and allow a fast and uneventful biological regeneration of bones.


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PATIENTS ACTIVITIES - INFLUENCE ON WEAR BEHAVIOUR OF TOTAL HIP ENDOPROSTHESES

Michael M. Morlock (1), Georg Bergmann (2), Matthias Honl (3)

1. Biomechanics Section, TUHH Hamburg University of Technology, Denickestrasse 15, 21073 Hamburg, Germany, [email protected]

2. Department of Orthopaedics, Free University of Berlin, Berlin, Germany 3. Department of Orthopaedics, General Hospital Eilbek, Hamburg, Germany

ABSTRACT Pre-clinical testing of artificial joint replacements has become an established and required method to prevent catastrophic failures in the patient. Several studies have analysed patient activities in order to define representative loading protocols (e.g. Morlock et al., 2001). Suggestions for test setups of different complexities have been made based on these data (Bergmann et al., 2002). However, most of these approaches rely on cyclic testing or testing of singular events like stumbling. Resting phases, microseperation, increased back swing loads are not yet considered, even so these aspects might play an important role for wear, loosening or failure of artificial joints (Roter et al., 2002; Butterfield et al., 2002, Nassutt et al., 2003). This issues might become even more important due to the revival of large diameter metal-on-metal bearings. In the future additional pre-clinical testing methods might have to be defined for specific bearing material combinations or prosthesis designs. A new test setup is currently established to investigate the effect of different parameters (resting duration; movement frequency; load level, lubrication fluid, diameter, use state, implant type) on friction (Fig. 1).

Figure 1: Friction testing set up on the material testing machine.

REFERENCES Bergmann et al, Inst. Mech. Eng., 2002. Butterfield et al, Trans ORS: 128, 2002.


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Morlock et al, J Biomech 34:873-881, 2001. Nassutt et al, CORR 407: 127-138, 2003. Roter et al, Trans ORS: 100, 2002. ACKNOWLEDGEMENTS This study was supported in part by the European Community, Contract No. SMT4-CT96-2076. NOTES:


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Rapid identification of muscle activation profiles via optimization with smooth profile patches

A.Kecskeméthy, D. Strobach University Duisburg-Essen, Lotharstraße 1, D-47048 Duisburg, Germany

email: {a.kecskemethy, d.strobach}@uni-duisburg.de web page: http:://www.mechanik.uni-duisburg.de

The identification of muscle activation profiles for general motions of the musculoskeletal system is an important and still open problem in biomechanics. Current methodology consists in finding the amplitude and the time history of the activation functions through optimization techniques. In this setting, function parameterizations such as discretization through sampling values, rectangular and ramp functions, as well as splines have been investigated. However, typical optimization runs display prohibitively long computation times, which make them unsuitable for on-line applications, such as required for example for patient-specific therapy. The present paper proposes a new method for parameterizing muscle activation profiles employing smooth (C∞) base functions. The idea is that, for the target approximation of dynamical motions, the detailed geometry of the activation profile is not significant, while its smoothness facilitates the search within the optimization procedure. The results show that, by applying these methods, the required CPU time can be reduced by factors of up to 10-50. We analyze the method for a flexion/extension two-joint subsystem of hip and knee, comprising only two pairs of antagonistic muscles, showing its sensitivity to (1) sampling time, (2) weighting factors and (3) the initial values. From these studies it becomes obvious that determination of muscle activations through optimization can be very sensitive to not carefully chosen parameterizations. Keywords: musculoskeletal biomechanics, dynamic simulation, optimization, Hill-type muscle model


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Investigation of mechanical compatibility of implants / endoprosthesis and bone tissue. Simulation of intelligent systems on the basis of movable cellular automaton method

1S.G. Psakhie, 1Ig.S. Konovalenko, 2A.V. Karlov, 1E.V. Shilko, 1A.Yu. Smolin

1Institute of Strength Physics and Materials Science, Tomsk, 634021, Russia 2 Center of Orthopaedy and Medical Materials Science, SB RAMS, Tomsk, Russia

E-mail: [email protected] Artificial joints (endoprosthesises of hip joints) are widely used in traumatology and

orthopaedy. In most cases the materials of artificial joints (usually metals) have sufficient characteristics of biochemical compatibility with bone tissue. At the same time very important problem of their biomechanical compatibility is still unsolved. Surgical removal of the femoral head with part of the femoral neck, local destruction of the spongy bone and replacement of the removed bone by the implant, which is not mechanically compatible in full measure with the bone tissue, result in considerable change of the strength, deformation properties and the overall behavior of the artificial hip joint in comparison with the natural one. The changes in the mechanical properties of the joint can result in damage generation in the bone tissue under some kinds of dynamic loading. It leads to resorption of bone, aseptic implant loosening and, as a result, to necessity of complex reoperation of the patient. One way to solve the problem of mechanical compatibility may lie in the development of orthopedic systems with intelligent mechanical response. Such systems change their strength and deformation properties depending on the environmental stress. “Intelligent” systems can greatly increase biomechanical compatibility of endoprosthesis and bone tissue, especially under loading which endanger implant anchoring.

In this connection the investigation of the mechanical interaction of the implant and the bone tissue under dynamic loading is one of the most interesting and important tasks

nowadays. Note, that the final goal of such research is to develop the structure of endoprosthesis that can function as similar to the original joint as possible.

One of the possible modifications of the endoprosthesis structure is the introducing specific inclusions into its most critical parts. Experimental and clinical investigation of functioning of the modified artificial joints is very complex, long-continued and expensive. Thus, computer-aided simulation of human joint with installed endoprosthesis having heterogeneous damping structure looks very perspective for solving the actual problems of traumatology and orthopaedy. The movable cellular automaton method (MCA) is one of the most perspective approaches in computational mechanics nowadays. Therefore, in the present work the investigation of the influence of structure modification of steel endoprosthesis on behavior of the hip joint under dynamic loading imitating action on the straightened leg was carried out using the MCA method.

Variety of modifications of the artificial joint structure were considered: introduction of soft damping inclusions having the shape of trapezium in the neck of endoprosthesis, coatication of damping layer on the stem of endoprosthesis and so on. Simulation results showed that a modification of endoprosthesis structure can greatly slow down or even prevent initiation and development of destroying cracks in the thigh-bone canal. For example, in the case of joint with the inclusion in the endoprosthesis neck the value of ultimate strain was 11-15% higher than one for the joint with original endoprosthesis.

The results showed that unique capabilities of the MCA method allow investigation of the mechanical behavior of human joints and other parts of human body under real very complex loading conditions. In addition, a possibility appears to design structure of endoprosthesises or other implants on the basis of the individual anatomic peculiarities of the patients.


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Nanocrystalline hydroxyapatite and apatite binding in bone: a solid-state NMR perspective

C. Jäger* (Federal Institute of Materials Research and Testing, Berlin)

T. Welzel, M. Epple (Inorganic Chemistry, University of Duisburg-Essen) N.S. Groom, M.J. Duer (Univ. Cambridge, U.K.)

Hydroxyapatite (HAp) is an important inorganic biomaterial. HAp plays a major role for the

biofunctionalization of e.g. metallic implants or organic matrices. Furthermore, the interaction between collagen and apatite in bone is responsible for its remarkable mechanical properties,

i.e. hardness combined with elasticity. Recently, specific attention was given to produce nanocrystalline HAp with a narrow crystal size distribution by precipitation from aqueous

solution.[1] If such materials are characterised by XRD and/or IR, they give the typical patterns of nanocrystalline apatite, i.e. the reflections and absorption lines are significantly

broadened. 31P-MAS NMR spectra show an apatite resonance at 2.3 ppm which is also asymmetrically broadened. Using high-end solid-state NMR experiments, it will demonstrated that these nanocrystals consist of a crystalline HAp core that contains about 40% of the total amount of phosphorus. In contrast, the rather thick surface areas consist of distorted CaHPO4- and Ca(H2PO4)2-like phases including much water. Quantitative numbers of the crystal core and distorted surface region sizes are presented including the number of P-OH groups and the amount of water. It is clear from these results that the chemical properties cannot be described by HAp only, although this is the only crystalline phase detected by XRD. Finally, the results of solid-state NMR investigations of a natural bone sample are presented. These data give the first experimental evidence that (i) predominantly glutamate residues are bound to the apatite-like surface with a δ-13C-

31P distance in the range 0.45-0.5 nm, (ii) no P-O-C bonds are present and (iii) that proline and hydroxyproline are not involved in binding. A binding model is presented. [1] T. Welzel, W. Meyer-Zaika, M. Epple, Continuous preparation of monodisperse

functionalised calcium phosphate nanoparticles with adjustable crystallinity, Chem. Commun. (2004) 1204-1205.


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Individual Biodegradable Implants for Cranial Bone Reconstruction

C. Schiller(1), M. Epple(1), M. Wehmöller(2), C. Rasche(3), H. Eufinger(3), S. Weihe(3)

(1) Institute of Inorganic Chemistry, University of Duisburg-Essen, Germany

(2) Institute of Production Systems, University of Bochum, Germany (3) Department of Oral and Maxillofacial Plastic Surgery, University of Bochum, Germany

Defects of the skull, resulting from trauma or tumor extraction, are normally reconstructed by autologous bone transplantation, PMMA bone cement or metal implants. However, these established methods have drawbacks like resorption of transplanted bone, inflammation, unphysiological heat conduction of metal implants, or a unsatisfactory geometric result of the reconstruction. Biodegradable implants would offer interesting advantages: compared to autologous transplants, they can be more easily adapted to the defect geometry, and in contrast to PMMA or metal implants, they can be replaced by newly grown bone. Additionally, the risk of a permanent foreign object in the body is avoided. The bony bridging of a defect requires a sufficient bone forming potential. In the case of cranial defects, bone forming cells can grow into the defect not only from surrounding bone but also from the dura mater. Taking into account these aspects, we developed a two-layer-structure for a resorbable cranial implant (Fig. 1). Suitable materials that fulfill the requirements in mechanical strength and biocompatibility were developed especially for these implants. We used composite materials of polylactide and calcium phosphate/calcium carbonate. The inorganic filling material neutralizes the acidic degradation products of the polymer (Fig. 2) and improves the biocompatibility. The manufacturing of the implants was realized by a two-step-process: hot compression molding of the compact outer layer and foaming of the porous inner layer with supercritical CO2. Individual moulds are used in both processing-steps (Fig. 3), planned and manufactured by CAD/CAM-methods on the basis of computer-tomography-data of the patient.

Figure 1: two-layer-concept for a cranial implant Figure 2: pH curves of composites immersed in water


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Figure 3: Steel mould for hot pressing of the compact layer (left), PTFE mould for foaming of the porous layer (middle) and the implant made in these moulds (right)

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Reconstruction of acute Rotator Cuff Injury with new Alloplastic Tendon axially fixed

within a Precoated Bone Screw: Experimental Repair Model in Sheep

Sellei, R. M.1; Weber, M. ²; Marx, R. ²; Erli, H. J.1

1 Department of Trauma Surgery, University Hospital of the University of Technology, Pauwelsstrasse 30, 52072 Aachen, Germany 2 Department of Prosthetic Dentistry, Section of Dental Materials, University of Technology, Pauwelsstrasse 30, 52072 Aachen, Germany Abstract Background: Synthetic materials can be used as allograft augmentation and replacement of traumatically injured ligaments and tendons. The fundamental problems are fatigue of material with rupture and deficiency in fixation technique as well as in biocompatibility. The objective of our study was to implant an alloplastic tendon with a newly developed bone screw for in vivo testing.

Methods: The rotator cuff model we used in our study is well established. The infraspinatus tendon (n=8) of one limb was cut close to the insertion and an artificial tendon tear of 1 cm of width was added. This acute rotator cuff tear was reconstructed with a pair of newly developed alloplastic PVDF tendons. Those were fixed in the humeral head by a bone screw in which the alloplastic tendon was axially inserted by a new adhesive technique. The non operated limb was used as a control group. An early group of two sheep were killed after four weeks. In the intermediate group after eight weeks four sheep and in the delayed group after sixteen weeks two sheep were killed. After explantation the infraspinatus tendon with the PVDF tendon and the bone screws were analysed macroscopically and histologically. Biopsy specimens were obtained from the muscle on both sides and were analysed for muscular atrophy. The infraspinatus tendon of the non teared and reconstructed side underwent post mortem a pull out test of a stitched PVDF tendon. Results: The operated sheep showed good postoperative results. All bone-tendon-muscle complexes showed macroscopically healed integrity at time of explantation. Histologically we found a continuous decrease of inflammatory infiltration and giant cells. There was no significant change of the diameter of muscle filaments compared with the other side. The absence of atrophy can be interpreted as a physiological gain of muscle function after the tear repair. The average force of the pull out test was 110 Newton (n=6). So the initial tensile strength of the reconstructed tendon should be strong enough to ensure the function of the infraspinatus. With the healing process this load bearing is taken over by the scar tissue. Conclusion: We developed a new alloplastic tendon which underwent a preclinical in vivo testing. The observed outcome showed promising results. Analysing the PVDF tendon within the tissue resulted in a good biocompatibility. Thus, we established an alloplastic tendon repair of the rotator cuff in a sheep model. Clinical Relevance: The reconstruction of rotator cuff injury is associated with a high rate of failure. In order to reduce the postoperative immobilisation in elderly and non co-operative patients our alloplastic material could be used because of its initial tensile strength.


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Biomaterials – Industrial Quest to Research Falko Schlottig, Synthes, Development Biomaterials, Eimattstrasse 3, CH 4436 Oberdorf

Musculoskeletal conditions are among the most costly illnesses to treat, consuming on average three percent of total gross domestic product in developed countries. From arthritis to osteoporosis, fractures to dislocations, musculoskeletal diagnoses represent the primary nonpsychological causes limiting activity in people of all ages worldwide1. Growth drivers in the orthopaedic and trauma market are demographic developments (ageing population), population behaviour (activities), population expectations (treatment standards) and new products ( new treatment methods, enhancement of clinical outcome, new technologies). Biomaterials are part of the new technology sector and an important driving force for the growing market, valued at over $1.4 billion in 2003, the expectations are over $3.4 billion by 20082. The applications of biomaterials are widely spread: from hyaluronic acid injections to substitute synovial fluid in the knee to allografts used in spinal fusions and in synthetic materials used as substitutes for bone grafts in different indications. Other very important developments in classic biomaterial are porous metals, new alloys and ceramics, in particular new manufacturing technologies. The medical industry has a clear demand based on clinical needs and indications, they are aiming to solve real clinical problems in cooperating with academic and clinical partners. New operation techniques, expanding indications for currently approved biomaterials, the advent of second-generation products, increased surgeon familiarity with the use and introduction of new biomaterials and cost sensitive technologies can help to solve existing clinical problems in line with the budget of the public health care systems. There is no need to find solutions for non-existing indications or solved problems excepting cost sensitivity or patent protection of the technologies. Most of the new product developments are based on years or decades of basic and applied research, e.g. growth factors. The industrialisation of results from basic research is very often a time and cost consuming process. There is growing concern that many of the new basic science discoveries made in recent years may not quickly yield more effective, more affordable, and safe medical products for patients. This is because the current medical product development path is becoming increasingly challenging, inefficient, and costly3. Important steps during industrialisation are proof of principle and proof of concept, know how transfer, up scaling and regulatory issues. It is required that research results should not be single shots and should be well documented and described in every step. Processes, analytical procedures and resulting data must be shown to be reproducible and should be independent from on-site findings. The biological performance of the new materials as well as any influence caused by a new technology has to be investigated. This statement in theory can be found in every biomaterial book or review but the practice is different. From the point of view of medical industry it is essential to show the biocompatibility of the developed materials or devices as early as possible, which means in the research phase. Furthermore not only the properties of the material and the device itself are important, but without a dedicated operation technique the new development will fail in the market. In conclusion - for medical technology-performance is measured in terms of product safety and effectiveness. Finally all the product safety and effectiveness data based on the research and industrialisation activities are required for submission of the documentation and the approval by the notified bodies and, consequently, are the precondition for a successful product on the market. 1 The Worldwide Orthopaedic Market – 2002-2003, November 2003; Knowledge Enterprise Inc. - The OrthoPeople 2 US Markets for Orthopedic Biomaterials 2003; Millennium Research Group 3 Innovation / Stagnation – Challenge and Opportunity on the Critical Path to New Medical Products, FDA March 2004; http://www.fda.gov/oc/initiatives/criticalpath/whitepaper.html


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Session: Laboratory and Computer Simulation in Soft Tissue especially Cardiology Jörg Schröder (Duisburg-Essen, Germany) Aspects of modelling and computer simulation of soft tissues. Applications to arterial walls Gerhard A. Holzapfel (Graz, Austria) Balloon angioplasty and stenting: Mechanical and clinical aspects James Moore (College Station, USA) The role of biomechanics in arterial reactions to stents T. Aleksyeyeva (Kiev, Ukraine) Various inorganic coatings for modification of the metal stents surface Oleg V. Shekera (Kiev, Ukraine) Thromboresistance fluorocontaining poly(urethaneurea)s: Synthesis, properties and application Andreas Meißner (Duisburg-Essen, Germany) Laboratory simulation of stent fatigue


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Balloon angioplasty and stenting: Mechanical and clinical aspects

G.A. HOLZAPFEL Graz University of Technology, Institute for Structural Analysis – Computational Biomechanics Schiesstattgasse 14-B, 8010-Graz, Austria; e-mail: [email protected]

Balloon angioplasty is a well-established interventional procedure which aims at reducing the severity of atherosclerotic stenoses. Unfortunately, although most of the treatments are performed with stenting nowadays, the procedure still can fail due to resistance to dilation, thrombosis and stress-induced tissue growth such as neointimal hyperplasia within the axial stent length. The latter biological process is triggered by inflammation, granulation and cellular proliferation, which is a consequence of the changed stress situation in the arterial wall. Nevertheless, balloon angioplasty with stenting improves the short-term success rate and reduces rates of restenosis in long term, and, consequently, the need for further interventions when compared to standard angioplasty procedures. The Restenosis Stent (REST) Study [1], e.g., compared coronary-artery stenting with balloon angioplasty for restenotic lesions after initial balloon angioplasty. The randomized study considered a total of 383 patients with restenosis after balloon angioplasty who were randomly assigned to Palmaz-Schatz stent placement or repeat (standard) balloon angioplasty. Angiographic restenosis (>50% follow-up diameter stenosis) was lower in stent-treated patients (18% versus 32% in balloon angioplasty-treated patients; p=0.03).

The proposed approach intends to predict the outcome of patient-specific angioplasty treatment on a computational basis [2]. We alter several parameters in order to get the change of the mechanical environment during dilation, and to find the optimal treatment for a particular stenosis type. The determination of an optimal treatment, however, requires a quantification of the changes (here only mechanical) in the arterial wall during inflation and stent deployment. Hence, we propose the following strategy: first, evaluate mechanical field quantities, which are based on nonlinear finite element analyses; second, evaluate indicators with the goal to replace the field quantities by scalars enabling the establishment of a “ranking” among the results. These indicators are derived numerically and allow a characterization of

the change of the intimal pressure caused by the struts of the stent, the stress change within the arterial wall caused by the stent, and the luminal change due to angioplasty.

The idea is to, somehow, minimize the changes of the intimal pressure and the arterial wall stresses by maximizing the lumen diameter increase for a particular stenosis.

The proposed approach allows to (i) analyze the three-dimensional interaction between stent models and patient-specific wall models by considering image data and mechanical tests performed at body temperature, (ii) quantify the change in the mechanical environment which occurs during stent placement, (iii) compare the effects of different parameters to identify optimal stent designs. Based on this approach stents may be parameterized such that novel designs and placement protocols are to be developed in order to minimize vascular injury and to optimize long-term success. It is important to note that the mechanical injury to the vessels is not eliminated with, e.g., drug-eluting stents so that the present study appears still meaningful in order to identify the biomechanical nature of stenting procedures. The methodology may eventually assist clinicians in choosing a particular stent with the goal to improve the clinical outcome.

Acknowledgements. Financial support for this research was provided by the Austrian Science Foundation under START-Award Y74-TEC.

References [1] R. Erbel, M. Haude, H.W. Hopp et al., Coronary-artery stenting compared with balloon angioplasty

for restenosis after initial balloon angioplasty. Restenosis stent study group, New Engl. J., 1998;339:1672-1678

[2] G.A. Holzapfel, M. Stadler, Changes in the mechanical environment of stenotic arteries during interaction with stents: computational assessment of parametric stent designs, J. Biomech. Engrg., in press


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The Role of Biomechanics in Arterial Reactions to Stents

James E. Moore Jr.

Biomedical Engineering Department, Texas A&M University, College Station, Texas, 77843-

3120, USA, [email protected] Restenosis in response to vascular stents may be related to biomechanical interactions with blood flow and the artery wall. Clinical studies have indicated that the risk of restenosis varies with stent design. However, the mechanisms through which stent design influences the artery wall response are not known. The effects of stent design on blood flow patterns arise from both compliance mismatch and strut geometry. In vitro flow studies have revealed large-scale flow disturbances and stagnation between the struts. The level of flow disturbance depends on the nature of the compliance mismatch, while the stagnation depends heavily on strut spacing. The degree of stagnation and its effects on wall shear stress affect platelet deposition, one of the early responses of the artery to the stent. Inflammatory responses are also known to be mediated by hemodynamics. Endothelial lining regrowth depends on flow patterns. In vitro studies indicate that the slowest regrowth occurs in areas of stagnant flow. The mechanical interaction between the stent and artery wall also results in a non-physiologic solid mechanical stress through the wall. Abnormally high wall stress concentrations have been predicted to occur at the ends of the stent. The stress on the artery wall, as well as the cyclic strain in the wall, depend heavily on stent mesh geometry. Our studies aim to understand the biomechanical interactions between the stent, blood flow, and the artery wall, and how they influence the physiologic reactions that lead to intimal hyperplasia and restenosis. Knowledge of the effects of stent design on biomechanical reactions of the artery wall will be used to improve stent design. Consideration of both hemodynamics and artery wall stress are crucial in this process.


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VARIOUS INORGANIC COATINGS FOR MODIFICATION OF THE METAL STENT’S SURFACE

Aleksyeyeva T.1, Lazarenko O.2, Homolyako I.3, Mikhalovska L4., Mikhalovsky S.4 1Institute of Physic Metals NAS of Ukraine, Kiev Ukraine

2Kiev’s Academy of postgraduate Education Ministry of Health of Ukraine, 3Institute of Surgery and Transplantology MAS Of Ukraine

4School of Pharmacy and Bimolecular Sciences, University of Brighton, UK

4-6 months after the stenting of coronary arteries in-stent restenosis occurs in 20-25% of the patients. A repeated operation is connected with significant financial expenses. A study was made of the response of the rabbit’s arterial wall to various inorganic stents coatings. Material and methods The 40 rabbits (male, breed “Grey Giant”, weight 2,7-3,0 kg) were selected for the experiment. The animals were maintained in individual cages at the temperature 24 ± 1ºС with a relative humidity amounting to 30-70% and 12/12 hours day/night cycle. The animals were kept at the standard diet with water ad libitum. In order to develop the rabbit’s model with a high atherogenic plasma, the animals were intramuscularly injected with a polysaccharide solution (1,25 mg in 2 days during 2 weeks). And after the operation, the animals received the injection of polysaccharide solution 1,25 mg/week during 4 weeks. Self-expandable Z-stents were made of 316L stainless steel. The diameter of wire was 0,2 –0,18 mm. The stents were implanted into the lumen of animal’s aorta under the intravenous anesthesia (thiopental solution 20mg/kg). The animals were divided into 5 groups of 8 animals according to the types of stents coating: amorphous carbon (AC), diamondlike carbon (DC), ceramic coating based on Titanium (Ti), Ceramic coating based on Zirconium (Zr) and a control group with stents of stainless steel (SS). In 8 weeks after the stenting, the animals were sacrificed and the aorta’s stented segments were collected for a histological study. The samples were histochemically studied by staining them with hematoxylin and eosin and by van Gison’s method. The morphometry was performed using the microscope Olympus BX-41 and DP-Soft. Results The morphometry data shows that in the case of AC, the neointima’s thickness was 2 times greater in comparison with the thickness of the intact vessel’s wall and with Zr’s group: 369µm/176µm – AC coating; 428µm/234µm – DC ones; 168µm/150µm –Ti; 208µm/µm 202 – Zr; 212µm/184µm – SS. Conclusion The use of ceramic coating based on Zr leads to the minimum neointima development over the stent’s struts. The study was made under the grant GR R31584/01 ‘Advanced biocompatible Endovascular stents’.


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;CH

3

2CH

CH O

n

;CH36C H3)( ;2СН

;( )CH2 4 On

=R2

R1 =

Thromboresistant Fluorocontaining Poly (urethane urea)s:

Synthesis, Properties and Application O.Shekera1, N.Kozak1, O.Lazarenko2, T.Alekseeva3, Yu.Nizelskii1

1Institute of Macromolecular Chemistry NAS of Ukraine, Kyiv, e-mail: [email protected] 2Kyiv Medical Academy of Post Graduate Education MAS of Ukraine, Kyiv 3Institute of Metal’s Physics NAS of Ukraine, Kyiv, e-mail: [email protected]

Important field of polymer materials application in cardiosurgery is manufacturing of

thromboresistant implants that function without vessel’s wall response on implantation. Synthesis and properties both biomedical and physic-chemical were investigated of

fluorocontaining poly (urethane urea)s (FPUU) used as coronary stent coating. The FPUU were synthesized in two step polycondensation method. First two series of linear polyurethanes were synthesized one based on toluene diisocyanate (2,4-, 2,6-TDI) and poly(propylene glycol) (PPG 1500) and another based on 4,4′-methylenebis(phenyl isocyanate) (MDI) and poly(tetramethylene glycol) (PTMG 1000). As polymer chain extender were used fluorocontaining diamines 4,4′-bis(p-aminophenylene)ester tetrafluorohydroquinone (p-BAPhETFQ) or 3,3′-bis(m-aminophenylene)ester tetrafluorohydroquinone (m-BAPhETFQ).

HH

NN C

OHH O

R R

O

N

H

NN N 32 1R1R CO

O

CC

Hn

,

=3

R

F F

O

FF

O ;

F F

F F

O O .

FPUU properties were analyzed using IR- and EPR- spectroscopy, thermogravimetry and

mechanical testing. Thromboresistancy of FPUU films was estimated via analysis of fibrinogen and platelet in model solutions and via determination of clotting parameters of whole blood sample after their contact with FPUU. Coronary stents with FPUU coating were implanted into rabbit abdominal aorta. The aorta wall morphology was analyzed gystologically.

FPUUs are segmented polymers soluble in DMFA, DMAA and other to form flexible films with excellent mechanic characteristics. The sensitivity was revealed of paramagnetic probe TEMPO rotational diffusion in FPUU matrix and FPUU thermooxidative destruction stages to presence of fluorine in polymer chain and to NH2-group isomeric position in the chain extender.

Thromboresistancy of FPUU is proved by retention of platelet functional activity under contact with the polymer. Morphological changes of aorta wall after stent removal are adequate in most cases to results obtained for animals control group. Therefore application of FPUU especially based on MDI,PTMG and m-BAPhETFQ is appropriate as vessel stent coating. Both thromboresistancy and aorta wall response depend on FPUU chemical nature


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and on NH2-group isomeric position in the chain extender (compare with EPR and TGA results).

Correlation was found between data obtained on investigation of the polymers structure - properties relationship using physical and chemical methods and data of the FPUU coating preclinical testing this correlation being useful in new biocompatible polymers development. NOTES:


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Laboratory Simulation of Stent Fatigue

Andreas Meißner, Alfons Fischer Mater. Sci. Eng. II, University of Duisburg-Essen, Lotharstr. 1, 47057 Duisburg, Germany,

[email protected] Since 1986 PTCA with implantation of a stent became a more and more important invasive method of treatment for coronary heart disease. The stent implanted during PTCA has to sustain a combined load of mechanical and chemical load. The pulsating artery leads to an expansion and cyclic mechanical load on the implanted stent. To eliminate the possibility of fail in vivo stents have to be tested regarding fatigue behaviour. As result of the complex geometry and the up to now not well investigated qualities of oligocrystalline structures /1/, so far fatigue properties of stents can only be carried out on the implants themselves. Additionally stent design plays a decisive role in terms of mechanical factors. Test equipment for fatigue tests on stents is offered e.g. by EnduraTEC (Minnetonka, USA). This equipment simulates human arteries by latex tubes. Cyclic pressure change inside the tubes is achieved by two electrodynamically driven actuators arranged at both ends of the tubes. The actuators are acting as fluid pumps. Expanding and contracting periodically in radial direction (caused by the pressure change inside the tubes) the latex tubes simulate conditions the stent is subjected to after implantation in the human artery. Inside the tubes Ringer’s Solution is used to simulate chemical load occurring in vivo. It has to be determined whether the stents show signs of mechanical fatigue and/or corrosion after a longer load period /2, 3/. Tests run at frequencies up to 50 Hz. After approx. 93 days, 400 million load cycles are achieved corresponding to 10 years of human life. Basing on the assumption that - in consequence of the pulse beat - a healthy human artery shows a cyclic expansion of 1 to 5 %, the control system of the test equipment is set to an expansion of 1 to 5 % of the latex tubes referring to the inner diameter. This expansion is detected beneath the stent implanted in the tube. As the direct measurement of the inner diameter isn’t possible the inner diameter of the latex tube has to be calculated on basis of the detected outer diameter. A new graded approach is presented and discussed allowing calculation of the inner diameter of a latex tube depending on duration of the test with contactless measurement of the outer diameter. A defined fatigue test of stents is enabled by well directed adjustment of the inner expansion of the latex tubes. /1/ Stolpmann, j.; Brauer, H.; Stracke, H.-J.; Erbel, R.; Fischer, A.: Practicability and

Limitations of Finite Element Simulation of the Dilatation Behaviour of Coronary Stents. MATWER 34 (2003) S.736-745

/2/ Guidance for the Submission of Research and Marketing Applications for Interventional Cardiology Devices: PTCA Catheters, Atherectomy Catheters, Lasers, Intravascular Stents. Draft Document, U.S. Department of Health and Human Services (1995)

/3/ Nichtaktive chirurgische Implantate. Besondere Anforderungen an Herz- und Gefäßimplantate. Spezielle Anforderungen an Arterienstents. Deutsche Fassung prEN 14299:2001. Januar 2002


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Session: Rapid and Virtual Prototyping in Medical Product Engineering and Therapy Planning Thomas Krell (Duisburg-Essen, Germany) Rapid product development of a critical care board for shortening the intensive – medical process chain Dieter Fichtner (Dresden, Germany) Reverse engineering and rapid prototyping for medical application Rudolf Meyer (Magdeburg, Germany) Rapid prototyping and reverse engineering for implants Barbara Leukers (Bonn, Germany) Biocompatibility of ceramic scaffolds for bone replacement made by 3D printing Wolfgang Freysinger (Innsbruck, Austria) Surgical navigation in space and time: what clinicians want, what is built, what they get. Is that what they need? Hans-Florian Zeilhofer (Basel, Switzerland) Computer-aided 3D-operation planning Ralf Schindel (St.Gallen, Switzerland) Biomaterials are going 3D with Layer Manufacturing Technologies (LM) – Direct and indirect approaches


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Rapid product development of a critical care board for shortening the intensive- medical process chain

Thomas Krell, Universität Duisburg-Essen, 45117 Essen, Germany

Rapid Product development (RPD) enables the possibility of using only digital product model data in the entire process chain of the complete product development. RPD uses a lot of technologies, e.g. :

• Computer-aided design, • Finite element analysis • Computerised numerical control • High speed cutting and • Rapid prototyping or tooling

Rapid product development has the following benefits:

• The number of real prototypes will be lowered • The costs will be reduced • The quality will be improved • The time to market will be shortened

The development of a stretcher systems is an example for rapid product development (RPD), as shown in figure 1. The system is named Critical Care Board (CCB).

Figure 1: Critical Care Board

The system is used for disabled people or inpatients, who are not able to move independently. The patients can be bedded on the system by the beginning of the treatment. They remain on the stretcher system during all diagnostic and treatment steps which includes also the shifting procedures, figure 2.


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Figure 2: shifting procedure with a sheet

A clinical evaluation period with functional prototypes showed, that the use of the system instead of using only a sheet (as shown in figure 2) has important advantages:

• Time is saved • The mechanical loads that affects the patient’s body with the shifting

procedure are reduced • The ergonomics of the shifting procedure is improved for the health

personnel • The integration into the existing medical equipment is easy possible

thereby at small costs. NOTES:


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Reverse Engineering and Rapid Prototyping for Medical Application

Prof. Dr.-Ing. habil. Dieter Fichtner, Dr.-Ing. Christine Schöne, Technische Universitaet Dresden, Faculty of Mechanical Engineering, Institute of Production Engineering

Prof. Dr.med.dent. habil. Bernd Reitemeier, Technische Universitaet Dresden, Medical Faculty „Carl Gustav Carus“, Department of Prosthetic Dentistry

Reverse Engineering and Rapid Prototyping can be used to change the manufacturing process of maxillofacial prostheses, individual repiratory masks, and extraoral radiation devices. They replace conventional impression materials such as silicones used until recently. During the conventional impression procedure soft tissues can be displaced. The displacement depends from the impression material and the method of positioning the patient [1]. Other methods to get a model of the body areas of interest – a virtual one – are the use of Computer Tomography (CT) and Magnetic Resonance Imaging (MRI) [2]. During this procedures the patient undergoes considerable exposure to radiation. Reverse Engineering and Rapid Prototyping avoid all disadvantages mentioned above. They are key elements of a process chain which begins with digitizing of extraoral defects and surrounding body areas by an optical 3D scanning device. Structured light will be projected on the body part of interest. The measurements should be done within seconds because a living person as object is involved in the procedure. An appropriate system was developed by the Fraunhofer Institute for Applied Optics and Precision Engineering IAO in Jena, Germany (see figure 1). Fig. 1: G-scanner (Source: FhG IOF) Fig. 2: Prosthesis pattern (orbital defect) During the measuring procedure data are recorded. Later they are transferred to a surface modelling computer system where a virtual model is generated. In the case of extraoral defects, the mirroring procedure is applied to model the virtual prosthesis pattern using the difference between the healthy and the diseased side (see figure 2). From that virtual model a physical prosthesis pattern is fabricated by means of a rapid prototyping method like ThermoJet Modelling. This pattern can be modified and corrected after evaluation at and by the patient. On the basis of the pattern the definitive orbital prosthesis is manufactured by the anaplastologist using the prosthetic material of choice. The same process chain (digitizing, surface modelling, Rapid Prototyping) can be applied in order to manufacture extroral radiation devices and individual respiratory masks. Reverse Engineering and Rapid Prototyping are steps in the production process of dental prosthetic restorations also [3].


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References [1] Öwall, B.; Käyser, A.F.; Carlsson, G.E.: Prosthodontics, Principles and management strategies.

London: Mosby-Wolfe, 1996, p. 201-221 [2] Penkner, K.; Santler, G.; Mayer, S.; Pierer, G.; Lorenzoni, M.: Fabricating auricular prosthesis

using three-dimensional soft tissue models. J. Prosthet. Dent. (1999) 82, p. 482-484 [3] Luthardt, R.; Weber, A.; Rudolph, H.; Schöne, C.; Quaas, S.; Walter, M.: Design and

Production of Dental Prosthetic Restorations: Basic Research on Dental CAD/CAM Technology. Int. J. of Computerized Dentistry (2002) 5, p. 165-176

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Rapid prototyping and reverse engineering for implants

R. Meyer, Magdeburg, Germany


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Biocompatibility of ceramic scaffolds for bone replacement made by 3D printing

Barbara Leukers1, Hülya Gülkan2, Stephan Irsen1, Stefan Milz3, Carsten Tille1, Hermann Seitz1,

Matthias Schieker2

1Research center caesar, Ludwig-Erhard-Allee 2, 53175 Bonn 2Chirurgische Klinik und Poliklinik – Innenstadt, Klinikum der LMU München,

Nussbaumstrasse 20, 80336 München 3Anatomische Anstalt, LMU München, Pettenkoferstr. 11, 80336 München

Bone grafts have been used for a long time to repair osseous defects from trauma or disease. While autografts are limited and difficult to shape, allo- or xenografts require extensive processing to minimize disease transmission. Today different synthetic bone replacement materials are available. Normally these materials are produced in simple geometries like blocks, pins or splines. Rapid Prototyping and especially 3D printing is well suited to generate bone grafts with more complex shapes as well as a designed internal pore network to mimic bone structures. These bone grafts are manufactured based on computer data. Furthermore, CT scan data of patients can be used to design an individual implant for a bone defect. 3D printing starts with a virtual dataset that is sliced by a computer and afterwards printed layer by layer on a ceramic powder-bed. After printing, the ceramic green part is sintered. We are able to produce matrices with a structural resolution below 500 µm after sintering.

We use synthetic spray-dried Hydroxyapatite (HA) granulates and a polymeric binder solution for the 3D printing process. The flexibility of this Rapid Prototyping technique allows investigation of different material combinations. The aim is to use the resulting ceramic matrices as scaffolds in tissue engineering. Therefore, it is important to investigate the cell ingrowth into the 3D matrix. To optimize the seeding efficiency, we developed two different designs of test bodies with defined internal structures. The objectives of both designs were maximization of the surface, facilitation of the seeding process and supply of the interior of the scaffolds with medium. In addition the mechanical stability of the test structures had to be sufficient.

To ensure biocompatibility of the ceramic test bodies, cell viability, cell proliferation and cell morphology were investigated according to ISO 10993-5 in static and dynamic cultivation setups. Metabolic activity of living cells was tested with a WST-1 assay kit. Additionally, the scaffolds were incorporated into Polymethylmethacrylate (PMMA) for histological evaluation. Cell adhesion on the designed internal structure, cell ingrowth into the bulk material and cell morphology were analyzed. An increase of metabolic activity and cell number was observed over a period of up to 7 days. No cytotoxic effects were noted. Especially in the samples cultivated under dynamic conditions cellular ingrowth into cavities at the surface could be detected.

These results show that the application of our newly designed ceramic scaffolds using 3D printing is promising for use as a bone replacement. In future, the osteogenic differentiation capability of human mesenchymal stem cells grown on these scaffolds will be investigated.


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Surgical Navigation in Space and Time: what clinicians what, what is built, and what they get. Is that what they need?

W. Freysinger, F. Kral, G. Diakov, Ö Güler, R. Stoffner

ENT Clinic, Medical University Innsbruck Anichstr. 35, A-6020 Innsbruck, Austria

Surgical navigation is a currently ever increasing technological approach to increase intraoperative safety by showing the surgeon the accurate position of an instrument inside the patient relative to some preoperative imagery. In general, preoperative CT scans with or without contrast agent are indicated in neurosurgical or oto-, rhino-, laryngological interventions at a typical slice thickness of 1 mm and less for microscopic interventions in the lateral skull base. All other standard radiologic imagery (MR, PET, SPECT, even ultrasound) can be used alternatively or can be fused to the CT-based imaging starting data sets. These are used in order to provide maximum reliability as provided by the high spatial fidelity of contemporary CT (multislice) machines and so can be used ideally to link the patient to his/her data in the preparatory phase. When superior functional or histological information is needed intraoperatively, MR or functional imaging is used for navigation. In order to achieve navigation to-day probes and pointers are placed at anatomical structures of the patient of some extrinsic marker structures and so the spatial coordinates of the patient can be linked to the preoperative data set. A variety of mathematical approaches and routines are implemented to perform this registration process. For the clinical user, no clue is given what the system is doing; it pops out a number, the root-mean-square-error, which is frequently cited as a quality measure. It may be an indicator that the registration was done well, but it need not. Ground truth is provided by direct verification on the patient. Once this step - the unsterile preoperative registration of the patient to his preoperative imagery - is successfully passed, the surgery proceeds as usual. So far, there do not appear specific requirements which could be much improved. But with sterile draping the situation changes dramatically, since this in the most cases destroys the established registration of the patient to his images. Based on our own intraoperative and clinical experience we address the following crucial points: is the current registration process the best one? What should the user-interface look like? How to interact with a computer in a sterile environment when the mind is loaded with responsibility for the patient? Why are navigated tools always cumbersome? Why is it that surgery / man has to adopt to technology and not the other way round? What can be done with respect of visualizing the actual position of the instrument in the patient? Is a flat monitor the best choice, what is with auto-stereoscopic displays, or with real three-dimensional environments, like the CAVE is? Are there no better ways to perform spatial digitizing to detect the patient and instruments? It will become clear that medical navigation is technology-driven and -dominated currently. It is, however, time to change this to create a knowledge-driven technological surgical paradigm which is the necessary prerequisite that high-tech enters "daily life". And only thereafter, the digital revolution in the operating rooms will continue and will positively affect further innovations. Acknowledgment: parts of this presentation were supported by the Austrian National Bank under grant No. 9318.


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Computer-aided 3D-Operation Planning H.F. Zeilhofer, Universitätsklinik für Wiederherstellende Chirurgie, Abteilung für Kiefer- und

Gesichtschirurgie, Kantonsspital Basel, Spitalstrasse 21, CH-4031 Basel In the last two decades craniofacial surgery has made an immense progress, which is based on new planning and simulating ways with computer-aided threedimensional technologies. It startet with new developments like rapid prototyping technologies. In the beginning the surgeon was handling with a non-transparent model of a patients skull, trying to shape on this model his operation lines. After the first promising results of planning with such an anatomical model the production of transparent stereolithographic models followed, which show not only the surface but also by color coding the sensibel and vulnerable inner structures of the skull. With 3D-model-guided simulations the planned osteotomy and bone rearrangement has become feasible. Important advantages of model based surgery: The surgeon is enabled to determinate the defect in comparison with the non-damaged area, to plan and optimize preoperatively complex craniofacial procedures, to anticipate the best operating pathway through sensible structures. The sterilized model even allows intraoperatively the modelling of individual templates or implants.

Picture 1 A color coded medical rapid prototyping model

Meanwhile the progress in computer-aided 3D-techniques and mathematical modelling allows to perform individualized treatment strategies which are planned in an integrated 3D-planning and virtual reality environment. Actual research results of our team in cooperation with groups in Berlin and Bonn are functional and dynamic treatment predictions in virtual reality, based on individuel FE-models. With those virtual 3D-models and a special software the surgeon can anticipate the behaviour of facial tissues with regard to natural jaw movement, surgical bone rearrangement, and muscle based mimics, even a smile.


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Picture 2 Virtual realy reprentation of the skull

In virtual reality-environment surgeons and other pecialists also can collaborate in distributed places. With a special hard- and software, a high-speed grid and video conferencing systems we are working on individual 3D-surgery planning and simulating. The result of such a collaborative distributed surgery planning can be presented the patient before undergoing surgery.

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Biomaterials are going 3D with Layer Manufacturing Technologies (LM) – Direct and Indirect approaches

RALF SCHINDEL1, GIDEON LEVY1, MANFRED ZINN2, CHRISTOPH LAMPERT3

1FHS University of Applied Sciences St.Gallen, Institute for Rapid Product Development, Tellstrasse 2, 9001 St.Gallen, Switzerland

2Swiss Federal Institute for Materials Testing and Research EMPA, Laboratory for Biopolymers, Lerchenfeldstrasse 5, 9014 St.Gallen, Switzerland 3CANTONAL HOSPITAL OF ST.GALLEN, Tumor Orthopedics, 9007 St.Gallen, Switzerland

In the last ten to fifteen years more then 30 different Layer Manufacturing Technologies (LM) based on different principles were developed. The most promising ones have been developed further and are used for prototypes, parts or models for presurgical planning - some fit into small market niches and some have just not survived. Two LM with a wide range of use, a big market segment and a high potential in R&D are the Selective Laser Sintering (SLS) – and the 3D Printing Processes. The main advantage of SLS is its big potential in processing materials to 3D parts. This is one reason why the Institute for Rapid Product Development (RPD) of the FHS University of Applied Sciences St.Gallen started 1996 with the SLS – Technology and the R&D of materials for the SLS process and runs today beside a 3D-Printing machine three SLS systems. Since that time two major materials where developed by RPD in close cooperation with the industry and are used now worldwide. A new SLS elastomer is in the product pipeline. The advantage of the 3D-Printing process is its resolution and its way to produce parts with model and support material. RPD has done many models of skulls for presurgical planning and different test geometries of bone structure and knows its potential in producing complex geometries as spongiosa. Some SLS tests have been carried out with biomaterials on the SLS machine and some geometry tests were done on the 3D-Printing system. This presentation gives an idea of the potential of the mentioned LM Technologies for the direct production of scaffolds in biomaterials. Out of a big range of different biomaterials RPD made trials with Polylactide (PLLA), Tricalcium Phosphate TCP and poly([R]-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB/HV). Spongiosa geometries have been 3D-printed in 4:1, 2:1 and 1:1 scales. The indirect LM production of an implantet bone substitute will be reported.


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Session: Biomaterials, Surfaces and Cell Interaction Markus Laub (Essen, Germany) 3D-rapid prototyping of protein molecules relevant for biomaterials Maria Chatzinikolaidou (Essen, Germany) Release kinetics of BMP-2 from metal implant surfaces Sven Henning (Halle-Wittenberg, Germany) Morphology, micromechanics and failure of bone cement Erwin Steinhauser (München, Germany) The osseous integration of RGD-peptide coated and non-coated metallic implants in the adult sheep Mark. R. Holt (London, United Kingdom) Measuring cell-substratum contact dynamics David Jones (Marburg, Germany) Stress sensing Adam Curtis (Glasgow, United Kingdom) Ionic controls of cell to cell interactions and membrane forces Lars Winkler (Essen, Germany) Cellular mobility on BMP covered surfaces Dietrich Büsselberg (Essen, Germany) Toxic effects of metals


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3D-Rapid Prototyping of Protein Molecules Relevant for Biomaterials

M. Laub and H.P. Jennissen

Universitaet Essen, Institut fuer Physiol. Chemie, Hufelandstr. 55, D-45122 Essen, Germany The highly conserved transforming growth factor beta superfamily (TGF-β) includes TGF-β1, -β2, -β3, growth differentiation factors, glial derived neurotrophic factors and 15 different different BMPs (bone morphogenetic proteins) [1]. These proteins have attracted a great deal of attention in biomaterial research due to their capability of controlling differentiation and proliferation in multicellular organism. Especially the unique ability of BMP-2 to induce ectopic cartilage and bone formation in vivo [2] has stimulated research in implantology. Recently we have described a novel application form for BMP-2, in which it is chemically immobilized on the implant surface. Implants prepared in this way have been shown to enhance bone growth and to accelerate integration [3-6]. Analyzing the molecular structure of these osteoinductive proteins may help to elucidate interactions between these molecules and the implant surface. Established techniques for depicting the molecular surface rely mainly on computational tools and the resulting two-dimensional representations of biomolecules are often hard to imagine in a three-dimensional way for the human beholder. In order to overcome these shortcomings we have developed a rather simplistic but highly efficient method for the construction of hands-on three-dimensional models of biomolecules. BMP-2 was one of the first molecules which was sculptured as three-dimensional polyamide model using the technique of 3D-Rapid Prototyping (3D-RP) (dimension 140 x 70 x 50 mm; 20 x 106-fold magnification) [7, 8]. Since these molecular sculptures are derived from X-ray structures they are remarkably exact replicas of the true molecules. Topographical characteristics become easily comprehensible in such a solid model whereas these features are barely visible in 2D computer screen models. In the meantime this technique has proven to be a valuable tool in the modeling of protein-protein-interactions as well as in the analysis of surface topography [7, 8]. Using these polyamide models we were recently able to identify a so far unknown structure on the concave side of bone morphogenetic protein 2 (BMP-2). This structure can be characterized as the imprint of a left-handed helix and was therefore called anthelix [1]. Further investigations using 3D-RP models of BMP-2, BMP-7 and TGF-ß2 demonstrated that the anthelix is found in all three dimeric proteins with similar values for pitch (ca. 8-9 nm) and radius (ca. 0.8-1.0 nm). These results were corroborated by measurements of the intersubunit angle of these dimeric proteins (141-149°) and the distances between the center of mass (1.68-1.96 nm) of the subunits both of which appear to be determinants of the anthelical pitch [9]. In contrast the anthelical motif was not found in dimeric molecules like human chorionic gonadotropin (hGC) which share an overall topological similarity of the monomers due to the so called cystine-knot motif but do not belong to the TGF-ß superfamily. Therefore the anthelical groove found in BMP-2, BMP-7 and TGF-β2 is probably a common structural motif of the members of the TGF-β superfamily. Since physiologically BMP-2 in bone tissue is found bound to matrix structures we speculate that the anthelix might be the binding site interacting with the matrix. We conclude that full scale 3D molecular models of biomolecules can lead to new perceptions in analyzing surface topography of biomolecules by macroscopic viewing and in-hand fitting of the molecules as demonstrated for TGF-ß-proteins. We therefore suggest utilizing such laser-sculptured macromolecules as a visual and tactile aid for enabling hands-on experience with macromolecules in research. 1. Hogan, B.L.M. (1996) Genes Develop. 10, 1580-1594 2. Urist, M.R. (1965) Science 150, 893-899 3. Jennissen, H.P., Zumbrink, T., Chatzinikolaidou, M., Steppuhn, J. (1999) Materialwiss.

Werkstofftech. 30, 838-845 4. Lichtinger, T. K., Mueller, R. T., Schuermann, N., Wiemann, M., Chatzinikoleidou, M., Jennissen,

H.P. (2001) Materialwiss. Werkstofftech. 32, 937-941 5. Voggenreiter, G., Hartl, K., Chatzinikolaidou, M., Rumpf, H.M., Jennissen, H.P. (2001) Materialwiss.

Werkstofftech. 32, 942-948 6. Jennissen, H. P., Chatzinikolaidou, M., Rumpf, H. M., Lichtinger, T., Mueller, R., (2000) In: DVM

Bericht 313, pp. 127-140. DVM, Berlin (ISSN 1615-2298) 7. Laub, M., Seul, T. Schmachtenberg, E., Jennissen, H.P. (2001) Materialwiss. Werkstofftech. 32,

926-930 8. Laub, M., Chatzinikolaidou, M., Rumpf, H., Jennissen, H.P. (2002) Materialwiss. Werkstofftech. 33,

729-737 9. Laub, M., Jennissen, H.P (2003) Materialwiss. Werkstofftech. 34, 1113-1119


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Release Kinetics of rhBMP-2 from Metal Implant Surfaces

Maria Chatzinikolaidou, Herbert P. Jennissen

Institut für Physiologische Chemie, Universitätsklinikum Essen, Hufelandstr. 55,

D-45122 Essen, Germany

Recently biologically active recombinant human bone morphogenetic protein 2 (rhBMP-2) has been chemically immobilized on titanium implant devices leading to enhanced bone growth and accelerated osseointegration in vivo [1-4]. Immobilization and desorption experiments are also carried out on cobalt chromium molybdenum alloy surfaces. To develop such a system the release of immobilized rhBMP-2 has to be controlled. In our procedure two methods of chemical immobilization of rhBMP-2 could be applied: covalently and non-covalently bound rhBMP-2. For non-covalent immobilization on chromosulfuric acid treated (CSA) metal surfaces rhBMP-2 was hydrophobically adsorbed to an alkyl substituted titanium surface of high alkyl surface concentration (Ti-CSA-APS). Covalent immobilization to the titanium surface (Ti-CSA-APS-CDI) was facilitated via silanization with 3-aminopropyltriethoxysilane (APS) and then coupling propylamino group with ε-amino group of lysine residue of protein by 1,1´-carbonyldiimidazole (CDI). In the latter case one obtains a mixture of covalently and non-covalently immobilized protein molecules, the latter adsorbing hydrophobically to the alkyl spacers of the silanized surface. Immobilized amounts of 125I-rhBMP-2 on titanium lie between 0.2-8.0 µg/cm2 of geometric surface depending on the texture. Release kinetics of 125I-rhBMP-2 on titanium and cobalt chromium molybdenum alloy surfaces provide us with important information for applying metal implants as drug delivery system in situ. Half lives were calculated according to a two-phase exponential model distinguishing between a fast phase at the beginning followed by a slow phase later on. Here we report on the slow final phase. In the case of a spontaneous release employing a physiological phosphate buffer saline at pH 7.4 approximately 90 % of the initial immobilized protein amount is released in a slow phase with half lives of 30 d for Ti-CSA-APS and 40 d for Ti-CSA-APS-CDI. In order to distinguish between covalently and non-covalently bound 125I-rhBMP-2, desorption by high concentrations of alkyl amine solutions at pH 7.4, e.g. with 2 M hexyl, heptyl and octyl amine is carried out. Additionally, the desorption by alkyl amine solutions serves as a proof for the mechanism of adsorbed rhBMP-2 by hydrophobic interaction. Latter method leads to a rapid release of 30 % of the initially immobilized protein amount within 6 h and the rest 70 % released from these surfaces with half lifes of 5-8 d for Ti-CSA-APS and 13 d for Ti-CSA-APS-CDI respectively. In contrast, ethyl amine only reduced the half lives to 18 d for Ti-CSA-APS and 28 d for Ti-CSA-APS-CDI. Similar half live values may be obtained on modified cobalt chromium molybdenum alloy samples. Therefore, we conclude a relation between half lives and carbon chain length of the alkyl amine solutions employed for the desorption indicating a stronger hydrophobic interaction by higher carbon chain length. Biologically active rhBMP-2 immobilized on metal surfaces could be measured by in situ activation of alkaline phosphatase in the mouse cell line MC3T3-M1 by fluorescence microscopy [5]. Cells grown on rhBMP-2 biocoated metal surfaces indicate in contrast to negative controls after 6 d a strong fluorescence as a proof of alkaline phosphatase activity. These results could be confirmed in vivo by employing a 1 mm gap-healing model in the distal femur condylus of sheep indicating complete osseointegration within 4 weeks. The measured half lives of 30-40 d correspond to the time required for physiological bone growth in vivo. From the desorption experiments on titanium plasma sprayed titanium alloy implants under consideration of the half lives of rhBMP-2 a theoretical release of ca. 80 ng/cm2 per day could be calculated. This illustrates the potential of bioactive implant surfaces for clinical applications.


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1. Jennissen, H. P., Chatzinikolaidou, M., Rumpf, H. M., Lichtinger, T. K., Müller, R. T., (2000) DVM Bericht 313, 127-140

2. Voggenreiter, G., Hartl, K., Assenmacher, S., Chatzinikolaidou, M., Rumpf, H. M. and Jennissen, H. P. (2001) Materialwiss. Werkstofftech. 32, 942-948

3. Jennissen, H.P (2002) Annals N. Y. Acad. Sci. 961, 139-142 4. Chatzinikolaidou, M, Laub, M., Rumpf, H., Jennissen, H.P. (2002) Materialwiss. Werkstofftech. 33,

720-727 5. Chatzinikolaidou, M., Zumbrink, T., Jennissen, H.P. (2003) Materialwiss. Werkstofftech. 34, 1106-1112

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Morphology, micromechanics and failure of bone cement

Sven Henning, Goerg Michler Institute of Materials Science, Martin Luther University Halle Wittenberg, D-06099 Halle/Saale,

Germany Email: [email protected]

Polymers for biomedical applications have to fulfill two main demands: Their surfaces should be compatible with respect to the living tissue that is in contact with the implant, and the mechanical properties should be suitable for the particular purpose. Needless to say that the material has to be nontoxic. Many of the applications require surfaces that exhibit a certain functionality (wettability, surface topography, tribology, functional groups, etc). On the other hand, mechanical compatibility of an orthopaedic implant (bone cements, plates, screws, etc) means a close match to mechanical parameters of the bone. Young´s Modulus of the implant should be close to that of the bone, and other properties have to fulfill certain minimal requirements (flexural strength, compressive strength, fatigue, wear, etc).

Transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM) were used to characterize the nanostructures of bone cements and bone before and after deformation. The interface between bone and bone cement was evaluated using ESEM. Additionally, energy dispersive X-ray analysis (EDX) was used to identify the inorganic components that are present in the composites.

Acrylic bone cements of different suppliers were investigated to identify critical defects that are responsible for premature failure of the cement that may result in implant loosening and, thereby, implant failure. The matrix polymer (polymethylmethacrylate, PMMA) can be modified by the addition of copolymers and inorganic particles, as there are antibiotics and X-ray opacifiers. A novel biocompatible and partially resorbable bone cement was prepared by the crosslinking copolymerization of oligo(lactone) methacrylate macromonomers and hydroxy ethyl methacrylate (HEMA) following a procedure that is described elsewhere [1-3]. The crosslinking reaction took place in presence of hydroxy apatite (HAp) of different origin and, thereby, of different particle size ranging from several 100 µm in agglomerates to few nm for finely distributed nanoparticles.

Morphology and micromechanical mechanisms that were observed for the composite materials are compared to the nanostructural and micromechanical features of human cortical bone. Bone can be defined as a hybrid nanocomposite material consisting of a polymer matrix (collagen) and inorganic nanoparticles (HAp). It has been shown that the resorbable bone cement that was under investigation stimulates the growth of new bone on the implant surface. In individual cases it was observed that bone is able to grow into pores and cracks that are near the sample surface. Matrix degradation may result in bone ingrowth. However, a good mechanical interlocking is at hand. The main disadvantage of this type of cement, the loss of mechanical strength after water uptake, was attributed to the existence of large agglomerates of the bioactive filler (hydroxy apatite, HAp). The incorporation of finely dispersed HAp nanoparticles yields a nanocomposite morphology that is similar to the nanostructure of compact bone.

[1] Seidel P, Sandner B, Gopp U, Schöch M, Steurich S, Santerre JP; Macromol. Symp. 144 (1999), 165-177

[2] Henning S, Adhikari R, Michler GH, Seidel P, Sandner B, Bernstein A, Hein H-J; in: Micro- and Nanostructures of Biological Systems, Shaker Verlag Aachen 2001, S. 109

[3] Helwig E, Sandner B, Gopp U, Vogt F, Wartewig S, Henning S; Biomaterials 22(19) (2001) 2695-2702


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The osseous integration of RGD-peptide coated and non-coated metallic implants in the adult sheep

E. Steinhauser1, T. Brill2, R. Bader3, S. Kerschbaumer1, A. Liebendörfer4

1) Klinik für Orthopädie und Sportorthopädie, Technische Universität München 2) Institut für Experimentelle Onkologie und Therapieforschung, TU München

3) Orthopädische Klinik und Poliklinik der Universität Rostock 4) Biomet Merck BioMaterials GmbH, Darmstadt

Introduction:

After the implantation of cementless endoprostheses an early osseous integration is strived for. Numerous in vitro studies could demonstrate that RGD-peptide coating can enhance the interaction between the surface of metallic implants and osteoblastic cells. However, only few information is available concerning the in vivo behaviour of such coatings. The aim of our study was to examine, whether RGD-peptide coating can improve the osseous integration of metallic implants in vivo. Materials and Methods:

After permission of the Animal Care and Use Committee an experimental study using 22 adult Merino sheep was carried out. Rough blasted metallic implants made of the Titanium alloy Ti6Al4V were examined with and without RGD-peptide coating. These implants were inserted in trabecular bone perpendicular to the axis of the bones by both, press-fit and defined gap fixation principles (maximum width 1.0 mm) in the left and right distal femora and proximal tibiae of the animals. After periods of 4 and 12 weeks mechanical pull-out testing and histomorphometrical analysis was carried out. Characteristic mechanical values (shear strength, interface stiffness, and energy absorption) were statistically analysed by the Wilcoxon Test. Results:

After 4 weeks there existed almost no differences between RGD coated and non-coated implants in the gap model fixation group. In contrast about 68% higher shear strength loads was measured for the RGD coated implants after press-fit anchorage (p = 0.071). Generally all characteristic mechanical values were higher at press-fit fixation in comparison to the gap model. After 12 weeks the differences between RGD coated and non-coated implants vanished. In contrast to the observations after 4 weeks there existed no longer differences between the two types of implant anchorage (press-fit vs. gap model). The histomorphological analysis revealed that RGD coated implants had improved osseous integration and less fibrous tissue ongrowth. Conclusion:

Even though the characteristic mechanical values of the interface metal-bone showed great variances and no statistical significance a 68% improvement in shear strength was evident for RGD-coated implants 4 weeks after surgery. Thus an advanced osseous integration of metallic implants due to RGD-peptide coating could be shown for the important early postoperative period.


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Measuring cell-substratum contact dynamics

M. R. Holt, London, United Kingdom


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High Resolution Optical Flow Measurement of Osteoblast Cell Traction Forces and Sensing of the Physical Environment.

David Jones, Sami Curze and Micah Dembo*

Experimental Orthopaedics and Biomechanics Pillips University Marburg and Dept.

Bioengineering Boston University USA In the 1970s Albert Harris showed, very elegantly, that fibroblasts apply considerable traction forces to their substrate. However for many years people still believed that healing wounds were drawn together by changes in the connective tissue collagen. In the last 8 years several methods have been developed to quantify and map these forces. One of these was developed my Micah Dembo, called optical flow traction force microscopy. Investigations showed several remarkably things. One was that cells seemed to use the tensions they apply to measure the stiffness of the substrate, and to orient themselves as to the substrate stiffness, crawling in the direction of stiffer substrates. This property is called durotaxis. Another interesting observation was that the forces did not seem to be generated in the so called stress fibres into the focal adhesions, but in the more diffuse actin cytoskeleton and nascent attachement sites. Our interest in using this technique was to measure how much force osteoblasts generate on stiff surfaces and how the cell regulated the tension when exposed to electrical fields or to mechanical perturbations

As in the figure above we can show that cells regulate their tensions with regard to the orientation of the applied electric field or mechanical stretch of fluid shear flow direction within in many cases a second and that this is not correlatable with intracellular calcium changes. The area of tension changes precedes also the areas in which the cells retract and also in the areas where the cells elongate, and thus must lay a part in the mechano-sensing apparatus of the cell. Other interesting observations include the measurement of tensions in osteosarcoma cells which are as a rule ten times weaker than primary cells, and that osteoblasts can apply forces equal to 2 million times their own weight.


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Ionic controls of cell to cell interactions and membrane forces

A.S.G.Curtis Center for Cell Engineering, University of Glasgow, United Kingdom

Membrane interactions controll cell adhesion and many other inter and intracellular events

and the roles of such events will be reviewed. Cations, in particular Na,K,and Ca, act in these

events by three different routes - as counter ions in electrostatic interactions of membranes, by

direct binding to various proteins, lipids and polysaccharides and by entry or exit from the cell

by ion channels involved thus in signal transduction. The relevant data on the relative

importance of these three routes will be discussed. These interactions will also be discussed in

terms of cell interactions with biomaterials.


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Cellular mobility on BMP-covered surfaces Lars Winkler, Institut für Physiologie, Universität Essen-Duisburg

Osteoblasts can move on the surface of implant materials as they do in osseous tissue. In general,

driving forces for cellular motility result from reversible binding of surface proteins (integrins,

cadherins) to extracellular matrix components (collagen, laminin), from re-arrangements of

cytoskeletal elements (actin, myosin), and also from local changes of the intracellular osmotic

pressure (e.g. by modulated activity of sodium-hydrogen-exchange). Bone morphogenetic protein-

2 has been demonstrated to stimulate cell motility in various cells, but the underlying mechanisms

remain obscure. Therefore, we studied BMP-2-mediated motility of osteoblastic MC3T3-E1 cells

in vitro.

Using time lapse video microscopy we were able to simultaneously record the movements of up

to several hundreds of cells under cell culture conditions (α-MEM medium with 1% fetal calf

serum, humidified atmosphere with 5% CO2 and 20% O2, 37oC). BMP-2 was provided either as

soluble protein, or as a BMP-2 depot (2-5 mm2) which was heat-immobilized onto a glass carrier

or implant material. Biological activity of free and heat-immobilized BMP-2 was assayed by

alkaline phosphatase (ALP) cytochemistry. A locally confined over-expression of ALP was found

where the morphogen had been deposited, suggesting that cells were stimulated by heat-

immobilised BMP-2. We further characterised the way how BMP-2 depots acts on cells and found

that ALP expression persisted even if the BMP-2 depots had been preincubated for up to 30 days

under cell culture conditions. In contrast, soluble BMP-2 lost its bioactivity within 3 days in cell

culture. On the other hand, heat-immobilized BMP-2 induced ALP expression also at remote sites

and further calculations led us to conclude that at least 3% of BMP-2 was released from a 100ng

BMP-2 depot within 3 days. Thus, heat-immobilized BMP-2 provides a combined source for

both, immobilised and diffusible BMP-2.

With respect to motility we found that the mean migration distance within 24 h of cells in BMP-2

supplemented medium and also on BMP-2 coated surfaces was increased (n=240) compared to

controls (uncoated glass, n=240). Most striking, cellular motility increased with increasing BMP-

2 concentration. In an attempt to identify molecular components whose expression might be

correlated with this increased motility, we analysed abundances of several mRNAs by RT-PCR

studies. Starting with most promising candidate genes we found that neither integrins (alpha 2, 5

and beta 1) nor cadherins (N-, OB-, E-, R-Cadherin) changed.

Together, the results show that soluble and heat-immobilized BMP-2 dose-dependently stimulates motility of MC3T3-E1 cells without changing the expression of the aforementioned molecules.


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Toxic effects of metals D. Büsselberg

Institut für Physiologie, Universitätsklinikum, Essen, Germany

Metals are able to shift physiologic to pathophysiologic processes, very often by interacting

with the calcium homeostasis of the cells. As calcium plays a major role in cellular function

the intracellular calcium concentration (Ca2+i) is tightly regulated. Interaction of metals in the

regulation of Ca2+i results in malfunction and regulated (apoptotic) or un-regulated (necrotic)

cell death. Possible sides of interaction for disturbance of the calcium homeostasis by metals

are: 1) membrane channels and/or 2) intracellular calcium stores.

1) Interaction at membrane channels

Voltage-activated calcium channels are most important gates through which calcium enters

neurones. Some metal ions, like Pb2+, Zn2+ or Al3+ are most effective in reducing the voltage-

activated calcium channel currents of mammalian neurones (cf. Büsselberg, 2004). For

example the threshold concentration for Pb2+ is about 0.1 µM.

Receptor activated channels are further potential sites of action for metals in the CNS. The

excitatory amino acid glutamate is important for processes such as learning and memory,

development and synaptic plasticity. Whereas activation of the glutamate and AMPA receptor

subtype mediates the fast excitatory postsynaptic potential (EPSP), the N-methyl-D-aspartate

(NMDA) receptor is involved in certain types of neuronal plasticity. Through the

glutamate/NMDA receptor channel complex, calcium enters neurones. Furthermore, the

NMDA receptor has modulatory binding sites for Mg2+ and Zn2+. A variety of studies have

provided evidence for an interaction of the NMDA receptor channel complex with metal

cations (Hubbarth et al., 1989; Ascher and Nowak, 1988; Uteshev et al., 1993). Pb2+ blocked

NMDA activated currents in both fast, reversible and slow, irreversible manners (Büsselberg

and Platt, 1994, Uteshev et al., 1993). The data indicate that glutamate receptors are

putative sites of action for metals. A metal-induced reduction of the channel current will

decrease the calcium rise in the neurones.

2) Interaction of metals with calcium stores

Some metal ions are capable to release calcium from the intracellular stores. This has been

shown for inorganic forms (Hg+) and some orgnic metals (e. g. methylmercury, trimethyltin;

Limke et al., 2003; Florea et al., submitted). While it has been proven for some metals that

only a small amount enters the cell (e.g. 0.5% for trimethyltin) and therefore could directly

interact with the receptor sites at the stores, the possibility has to be taken into account that

the calcium release might be also triggered by metal actions at the cellular membrane.


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Possible consequences: Calcium signals are vital for the survival of the single cell but also

for the communication between different cells. Therefore a change of the calcium signalling

not only results in the death of single cells but also changes higher functions. In regard to

the nervous system it has been shown that the long-term potentiation (LTP; see Bliss and

Lomo, 1973) of the synaptic transmission is impaired by some metals (e.g. Pb2+; Hori et al.,

1993). This could be a possible explanation for the learning and behavioural deficits which

were directly correlated to the blood levels of lead (Needleman, 1979).

Ascher P, Nowak L (1988). The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture. J Physiol

Lond 399:247-266

Bliss TV, Lomo T (1973). Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of

the perforant path. J Physiol 232(2):331-56

Büsselberg D (2004). Actions of Metals on Membrane Channels, Calcium Homeostasis and Synaptic Plastivity. In: Organometal and Metalloid

Specism in the Environment: Analysis, Distribution, Processes and Toxicological Evaluation; A. V. Hirner & H. Emons (Eds.), Springer, Wien,

New York, 259-281

Büsselberg D, Platt B (1994). Pb2+ reduces voltage- and NMDA-activated calcium channel currents. Cell Molec Neurobiol 14:711-722

Florea A-M, Dopp E, Büsselberg D (submitted). Elevated calcium transients by trimehtyltin chloride in HeLa cells: types and levels of elevation.

EHP.

Hori N, Büsselberg D, Matthews MR, Parsons PJ, Carpenter DO (1993). Lead blocks LTP by an action not at NMDA receptors. Exp Neurol 119:192-

197

Hubbarth CM, Redpath GT, MacDonald TL, VandenBerg SR (1989). Modulatory effects of aluminum, calcium, lithium, magnesium, and zinc ions on

[3H]MK-801 binding in human cerebral cortex. Brain Res 486(1):170-4

Limke TL, Otero-Montanez JK, Atchison WD (2003). Evidence for interactions between intracellular calcium stores during methylmercury-induced

intracellular calcium dysregulation in rat cerebellar granule neurons. J Pharmacol Exp Ther., 304:949-58

Needleman HL, Gunnoe C, Leviton A, Reed R, Peresie H, Maher C, Barrett P (1979). Deficits in psychologic and classroom performance of children

with elevated dentine lead levels. N Engl J Med 300:689-695

Uteshev V, Büsselberg D, Haas HL (1993). Pb2+ modulates the NMDA-receptor-channel complex. Naunyn Schmiedebergs Arch Pharmacol

347:209-213

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Postersession (alphabetical order) M. Albulescu (Bucharest, Romania) Polifin – a polymer that can be used as a drug and a controlled drug release system T.A. Alekseyeyva (Kiev, Ukraine) Exfoliated Grapite as Adsorbtive Material for Biological Application I. Bolle (Duisburg-Essen, Germany) Effects of hydroxylapatite-enriched bone cement polymethylmethacrylate on bone healing in the Yucatan swine J. R. Dabrowski (Bialystok, Poland) Metal-bioglass composite as a new biomaterial for bone surgery L. Denisyuk (Kiev, Ukraine) New Adpative Composition for Keratoplasty A.-M. Florea (Duisburg-Essen, Germany) Modulation of intracellular calcium by trimethyltin chloride in human tumor cells: neuroblastoma SY5Y and cervix adenocarcinoma HeLa S3. M. Guggenbichler (Erlangen, Germany) Development of a Ceramic Port Catheter with Antimicrobial Activity and Improved Fluid Dynamical Properties F. Hauschild (Jena, Germany) Establishment and validation of a biocompatibility assay for magnetic nanoparticles B. Hoffmann (Bayreuth, Germany) Influence of surface roughness and biofunctionalisation on the cell behaviour of implant material F. von Knoch (Chur, Switzerland) THE EFFECT OF SIMVASTATIN ON POLYETHYLENE PARTICLE-INDUCED OSTEOLYSIS N. Kozak (Kiev, Ukraine) Tuberculostatic Hydrazides of Isonicotinic Acid: Isoniazide, Propioazide, Ftivazidum and Flurenizide. Quantum Mechanical Simulation. D. Lattner (Duisburg-Essen, Germany) Reaction Analysis of Chromosulfuric Acid Action on Transition Metals at High Temperatures H.-G. Lipinski (Dortmund, Germany) Tissue deformation modelling: measurements and simulation S. Magosch (Dortmund, Germany) A computerized method to analyse bone cell mobility in vitro V. F. Pichugin (Tomsk, Russia) Deposition of calcium-phosphate layers on the materials of medical implants by magnetron sputtering


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M. Pietraszczyk (Jena, Germany) An improved 96-well microplate assay to analyze osteogenic activity of bone morphogenetic proteins O. Prymak (Duisburg-Essen, Germany) Coating of nickel-titanium shape-memory alloys with calcium phosphate by plasma-spraying O. Prymak (Duisburg-Essen, Germany) Fatigue of orthodontic Nickel-Titanium (NiTi) wires in different fluids under constant mechanical stress S. Reuter (Duisburg-Essen, Germany) Correlation of Structural Properties of Commercial DLC Coatings to their Tribological Performance in Biomedical Applications T. Ricken (Duisburg-Essen, Germany) Modeling of growth processes in soft tissues A. Roth (Dortmund, Germany) Sharpening and de-noising algorithms to enhance the quality of digital confocal colour images of cultured vital bone cells G. Saxler (Duisburg-Essen, Germany) Localization of SP- and CGRP-Immunopositive Nerve Fibres and of the NK1-Receptor in the Hip Joint of Patients with Femoral Neck Fracture, Osteoarthritis and Failed Hip Implants. I. Schmitz (Bochum, Germany) Influence of Condensation Technique on Quality of Direct Gold Restorations V. Sokolova (Duisburg-Essen, Germany) Transfection of cells with custom-made calcium phosphate nanoparticles K. Suck (Hannover, Germany) Multifunctional Polymer-Mineral Composite Materials as Bioactive Scaffolds for Bone Tissue Engineering V.K. Tsuber (Minsk, Belarus) BIOMATERIALS AND OPHTALMOLOGICAL IMPLANTS BASED ON NANOCRYSTALLINE HYDROXYAPATITE C. Wedemeyer (Duisburg-Essen, Germany) A SINGLE DOSE OF ZOLEDRONIC ACID MARKEDLY DECREASES PARTICLE-INDUCED BONE RESORPTION C. Wedemeyer (Duisburg-Essen, Germany) EXTENSIVE REGIONAL BONE FORMATION IN A MODEL OF PARTICLE INDUCED OSTEOLYSIS AFTER TREATMENT WITH ZOLEDRONIC ACID K. Ch. Wierzcholski (Gdansk, Poland) Intelligent Bioreactor for Tissue Cultivation


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Polifin – a polymer that can be used as a drug and a controlled drug release system Mihaela Albulescu, R. Albulescu, Gh. Seitan

National Institute for Chemical-Pharmaceutical R&D, Bucharest, Romania Calea Vitan 112, Sect. 3, tel: +0421 322 2916, fax: +4021 322 2917. e-mail: [email protected]

Introduction Polifin is the generic name for cross-linked hydrogels, obtained by radioinduced polymerization of acrylic acid. Similar, non-reticulated products are Carbopol and Carbomer. The reticulation of Polifin S is responsible for its ability to swell in water and its character of permanent hydrogel. The aspect of the product is a white powder, odorless, with a great swelling capacity (> 300 x). The main advantages of the product consist in its high purity, lack of residual polymerisation catalysts, the control of reticulation index, and the possibility to include various thermosensitive products in the polymerisation mixture, generating various matrices for controlled release of active compounds. Materials and methods POLIFIN (P) was tested as a drug: - haemostatic effect (test on rabbit ears) - cicatrisation effect in burns and cutting wounds (test on experimental wounds in rabbits) - cytotoxicity in hepatocytes culture POLIFIN tested as a controlled drug delivery system: With crystal violet (PV): - antimicrobial activity - haemostatic effect - effect in cicatrisation (experimental wounds in rabbits and 2 case studies: an infected wound two weeks old and a venous ulcer at a diabetic patient, treated for 7 years with classical drugs) With antibiotics: Sodium oxacilin Streptomycin sulphate Results and discussions Antimicrobial activity depends on the test-microorganism, crystal violet concentration and releasing time (fig 1-6) 1

2 3

4

5 6

The haemostatic effect Cytotoxicity of Polifin of P and PV in hepatocytes culture

Hemostatic effect of P and PV

0

50

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1

Group

Hem

osta

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(% fr

omco

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polifin

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Histology of wounds: A) cutting wounds

control

P

PV B) experimental burns

control

P

PV Effect of PV in the treatment of infected

wounds abdominal infected wound, 2 weeks old

4 days after a single application of PV

Effect of PV in the treatment of venous ulcer at a diabetic patient

7 years old venous ulcer treated with

classical drugs

after 4 weeks of treatment with PV

Release of antibiotics from Polifin matrix Release of Oxacilin from Polifin

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reactions, as well as good potential for applications in the treatment of wounds of various ethiology and in surgery. Also, some studies have demonstrated interesting properties of Polifin as carriers for various drugs, such as antibiotics, disinfectants or antalgic substances.

Thus, a wide area of potential clinical application is open for the product, and

clinical trials are expected to be carried on.

References: 1 Meredith M.J. - Cell Bioll. Toxicol. 4, (4) 405,1988 2. Ponta C. Seitan Gh. et al., Proc. Intl. Biomed. Eng. Days, Istanbul, IEEE 1992 464-467


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EXFOLIATED GRAPHITE AS ADSORBTIVE MATERIAL FOR BIOLOGICAL APPLICATION

Alekseyeyva T.A1., Sementsov Yu.I3., Lazarenko O.N. 2, Yanchenko V.V. 3

1Institute of Physic of Metals NAS of Ukraine, 2Kiev’s Medical Academy of Postgraduate Education of Ministry of Health of Ukraine, Kiev

3TMSpetsmash Ltd Kiev, Ukraine.

Exfoliated graphite is well known as a good material for the adsorption of the hydrophobic liquids. It was interesting to apply these properties biological purposes.

Materials and methods

A study was made of adsorption properties of exfoliated graphite that was obtained by different methods. The chemical and physical properties are represented on table 1. The samples of exfoliated graphite were soaked in physiologic solution 0,9% of NaCl and after that they were dispensed in the volume of 1 ml into the 24 wells plate. Then the liquid was replaced with fresh human’s plasma in 1:1 ratio of solid/liquid.. After a 4 hours exposure at 25 0C of the exfoliated graphite with plasma, the samples of plasma were collected for the further biochemical analysis. A biochemical study was carried out with automatic biochemical analyzer and kits BioSystem (Spain). The exfoliated graphite adsorption properties were evaluated by decreasing the level of the Cholesterol, Triglycerides, Glucose, Albumin, Urea, Creatinine, Total Protein and Electrolytes in comparison with the original plasma.

Results In comparison with the common carbons sorbents as SKN, KAU, the exfoliated graphite shows a good ability to adsorb biological molecules such as Urea, Creatinine (25% and 30% vs 3% and 12%), and a mild ability for ion-exchange properties for K+ (25% vs 80%) Conclusion The exfoliated graphite is a prospective material for the adsorption of biological liquids. Contact person Dr. Yu. Sementsov e-mail: [email protected]


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Effects of hydroxylapatite-enriched bone cement polymethylmethacrylate on bone

healing in the Yucatan swine

I. Bolle

Central Animal Laboratory, University Duisburg-Essen Medical Shool

Hufelandstr. 55, 45122 D-Essen

The subject of this experimental study was to test the integration ability and compatibility of two improved bone cements in comparison with Palacos® in the Yucatan swine. Bone cement A consists of polymethylmethacrylate, methylmethacrylate, n-butylmethacrylate, benzoylperoxide, dicyclohexylphthalate and up to 20% hydroxylapatite. Bone cement B contains polyethylmethacrylate, benzoylperoxide and up to approx. 40 % hydroxylapatite. Bone cement C was (Palacos®). The monomer for cements A and C consists of methylmethacrylate stabilized using hydroquinone and N, N-dimethyl-p-toluidine. On the other hand, the monomer for bone cement B contains n-butylmethylacrylate and N, N-dimethyl-p-toluidine. The animals were divided into four groups of six females each. All pigs of each group were operated on the same day. The bone cements were distributed by chance using a randomized scheme so that all the cements were implanted into the left and right femur. The knee joint was opened medially under anaesthesia and the patella was luxated laterally, allowing access to the facies patellaris. A defect in the cartilage and the subchondral bone was cut in the fossa intercondylaris using the Diamond Bone Cutting System (Fa Synthes) and filled using the cement, which was mixed under vacuum. Finally, the patella was replaced and the knee joint closed by surgery. After implantation animals were observed for 5, 10, 20 weeks, and one year. Any signs of lameness during the observation period were documented. Finally, the pigs were anaesthetized and blood replacement was effected with modified Karnovsky's fixative. Towards the end of this procedure, the pigs were euthanized.

Histological bone preparations were made by the separate thin section technique, developed by Donath (1997/98). They were dyed using toluidine blue to allow analysis of the cellular reaction between bone and implant and bone and cartilage formation at the interface. With regards to the bone contact at the interface, the best results were obtained using bone cement A (polymethylmethacrylate plus 20% hydroxylapatite). This applied to all groups (5 to 52 weeks). A thick layer of connecting tissue was only apparent after 5 and 10 weeks. After 20 or 52 weeks, in most areas of this layer had already become very thin, with frequent interruptions. The second best results were obtained with cement C (pure polymethyl methacrylate). The percentage share of direct contact between bone and cement was significantly lower than with cement A. Nevertheless, specimens with direct contact were found in all the groups. In almost all cases, the layer of connecting tissue was considerably thicker, with fewer interruptions than in the case of cement A. The poorest results were obtained with cement B. After 5 and 10 weeks no contact between bone and cement was observed. After 20 weeks, significant contact was only apparent in one of 6 cases. Large areas of contact were found in three of the animals observed for one year. Cement A is recommended for clinical application, whereas cement B is unsuitable.


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Metal-bioglass composite as a new biomaterial for bone surgery

Authors: Jan R. Dabrowski, Zbigniew Oksiuta

This work presents the analysis of research to determine influence of bioglass(1) (15%CaO, 9%P2O5,

76%SiO2) addition (0%, 5%, 10%, 15% wt.) on the structural, mechanical and corrosion properties of sintered samples based on Co-Cr-Mo alloy. Cylindrical samples φ20×10mm were prepared according to the following scheme(2): 1) blending of Co-Cr-Mo powder alloy with bioglass addition of 5%, 10%, 15%wt. in a ball miller, 2) uniaxial cold compaction at pressure of 600MPa, 3) sintering in the horizontal tube furnace at the temperature 1230°C in a dry argon atmosphere, for 1 hour,

4) rotary cold repressing on a PXW 100A press at pressure of 600MPa, number of rotations i=12, with spinning upper tool movement, 5) heat treatment at the temperature of 1230°C for 1 hour in argon atmosphere, cooling in the cold area of the

furnace. The microstructure of samples after heat treatment exhibited homogeneous distribution of bioglass particles

(average size 100 microns) in the Co-Cr-Mo matrix with an average grain size 40 microns(3) (Fig.1).

a) b) Fig. 1. SEM of samples before corrosion investigations: a) 5%wt. bioglass, b) 10%wt. bioglass

The compressing tests has shown (Table 1) that an addition to the Co-Cr-Mo matrix 5%, 10%wt. of bioglass

caused an increase of the yield strength (from 495MPa to 610MPa for 10% wt. of bioglass), but decrease of UCS and plastic strain of specimens. The higher corrosion resistance was obtained in the case of the samples with bioglass addition. With the increase of the bioglass addition, the corrosion resistance also increases. The microscopic examination after the potentiodynamic test revealed a dual character of the samples surface. The samples made with a pure Co-Cr-Mo alloy have vast area covered with pits with different shape and depths. The impedance tests confirmed the potentiodynamic corrosion investigation. The reaction resistance value of the samples with bioglass addition is over 50 times higher than that of the pure porous Co-Cr-Mo alloy.

- Table 1. Mechanical properties of composite samples after heat treatment (1230°C, 1h)

Materials UCS, MPa YS0.2, MPa Contraction, % HV0.1 Density, % 0% bioglass 5% bioglass 10% bioglass 15% bioglass

1580.0±52.6 1438.9±48.3 1124.3±23.4 795.2±44.6

456.0±44.9 590.8±20.1 610.3±22.2 536.0±34.5

27.6±2.3 19.8±0.9 13.0±1.2 5.8±1.4

334±36.5 390±21.0 457±30.2 519±34.5

87.0 88.3 84.2 79.9

* Average value of 5 samples of each material

Thus, from the mechanical and corrosion point of view, the composites with 10% of bioglass addition can be suitable material for further biological investigation.

- References:

1. Łączka M., Cholewa K., Łączka-Osyczka A.: Gel-derived powders of CaO-P2O5-SiO2 system as a starting materials to production of bioactive ceramics. J.All. Comp., 148 (1997), pp.42-51.

2. Oksiuta Z., Dąbrowski J.R.: Rotary cold repressing and heat treatment of sintered materials from Co-Cr-Mo alloy powder. Powder Metallurgy, 45 (2002),pp. 63-66.

3. Oksiuta Z., Dabrowski J.R., Olszyna A.: Co-Cr-Mo alloy-based biocomposites with bioactive glass additive, J. Mat. Process. Techn. (in printing).


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NEW ADAPTIVE COMPOSITION FOR KERATOPLASTY (Experimental study)

Denisyuk L.2,Denisyuk N.2, Aleksyeyeva T.1, Yanchenko V.3 1Institute of Physic Metals NAS of Ukraine, Kiev Ukraine

2 Center of Eye Microsurgery of Ukraine, Kiev, Ukraine 3 BÉRCRE KFT, Budapest, Hungary

Keratoplasty is a successful method for eyesight restoration in the patients with cornea disease. Keratoplasty is connected with the use of donor material and may result in rejection reaction, which is the main problem of modern ophthalmology. The repeated operation is connected with the use of the donor’s cornea, and any improvements in the method are encouraged. A new pharmaceutical composition was used in the study to increase adaptive properties of the donor’s cornea to the recipient’s tissue. Company BÉRCRE KFT (Hungary) has courteosly offered the composition for our research. The adaptive properties of composition for increasing the viability of donor’s cornea after keratoplasty were studied. Material and methods The 8 rabbits, (male, breed “Grey Giant”, weight 3,0-3,5 kg) were selected for the experiment. Keratoplasty was performed under the complex anaesthesia (intramuscular injection of ketamine 0,5ml/kg and intravenous injection of thiopental solution 20 mg/kg). After the separation, the donor cornea was put into the bathtub with the adaptive composition for 10-30 minutes and than implanted onto the eye of the rabbit-recipient. In order to minimize the amount of animals in the experiment and to receive the objective results, every experimental animal was a donor for the following one. The donor’s cornea was fixed by single knotty stitches by the periphery of the circle (suture 11.0 Cobra Ethycon UK). In the course 5 days, the animals were treated with eye-drops “Luxomicin” (Tubilux Italy)- 3 drops 2 times a day. The engraftment of the donor’s cornea was evaluated by its transparency and reflectivity using a slit lamp Results The operated rabbits were observed on the 3d, 10th, 24th and 80th days. The donor’s corneas were transparent and reflective in 7 rabbits. One animal had a dimmed cornea due to an incomplete match between the donor’s material and the recipient tissue. During the study no animals perished. Conclusion The new adaptive composition could be used for keratoplasty in order to significantly raise the probability of the viability of donor’ material.


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Modulation of intracellular calcium by trimethyltin chloride in human tumor cells:

neuroblastoma SY5Y and cervix adenocarcinoma HeLa S3.

A.-M. Florea1, F. Splettstoesser2, E. Dopp1, D. Büsselberg2

1. Institute of Hygiene and Occupational Medicine, University Hospital Essen, Germany 2. Institute of Physiology, University Hospital Essen, Germany

Environmentally relevant volatile organometallic species such as alkyltin with short carbon-chains are highly toxic. Trimethyltin (TMT) chloride is industrially produced and released into the environment. TMT was reported as neurotoxic, cytotoxic and genotoxic. Moreover, human intoxication migth result in severe health complications but possible toxic mechanisms are still unclear. It was suggested that the site of action for organotin compounds may be at the cellular membrane as well as in the intracellular space. While it has been shown that other organotin compounds increase cytosolic free Ca2+, data for trimethyltin are limited. Previously it was shown that modifications in intracellular Ca2+ concentration are involved in cellular signalling (e.g. neurotransmitter release, synaptic plasticity, muscular contraction, cell proliferation, gene expression) and cell death (apoptosis, necrosis). In this study we used laser-scanning microscopy in neuroblastoma and HeLa S3 cells loaded with fluo 4, to monitor dynamic changes of cytosolic calcium (Ca2+

i) in the presence of trimethyltin (TMT). Concentrations of 0.25 µM to 500 µM TMT were applied in Tyrodes buffer with a flow application system. TMT induced elevations in cytosolic calcium of neuroblastoma and HeLa cells. In HeLa S3 cells calcium increased in the following order: 0.5 µM ~125.6 %; 5 µM ~137.5 %; 500 µM ~202.5 %. In neuroblastoma cells the increase was: 0.5 µM ~133.3 %; 5 µM ~136.1 %; 500 µM ~147.1 %. This effect was reversible and a second application resulted in a similar rise of Ca2+

i when compared with the first application. Ca2+i was released from internal

stores, since there was no significant difference in the elevation of Ca2+i when an external

solution with no added Ca2+ was used (145 ± 15 % compared to 151 ± 10 %; 500 µM TMT). This difference was similar in neuroblastoma cells, were ~127 vs. ~ 136 % increase (5 µM TMT) was measured. In addition, after releasing calcium from the internal stores by 10 mM caffeine - before applying TMT (500 µM) - the rise of Ca2+

i was highly reduced (< 10 % increase) underlining the previous conclusion. This is not in line with the results from Ade and co-workers. While they also demonstrated that TMT, evoked an increase of the cytosolic free calcium concentration in human HL-60 cells they concluded that this effect was mainly due to an influx from the extracellular space and Ca2+ entered the cell via opened calcium channels (Ade et al., 2003). Interestingly, as a result of dose response, the number of reacting cells was directly correlated to the concentration of TMT used. While all cells reacted with 500 µM, only 80 % of the cells showed an increase with 50 µM and this percentage decreased further to 74 % with 5 µM. Finally, with all concentrations of TMT used, fast Ca2+

i transients (spikes) were observed, which also did not depend on the calcium concentration of the external solution. Our results suggest that TMT influences Ca2+

i homeostasis of HeLa S3 cells as well as of neuroblastoma cells. However this observation appeared to be different when compared with other cell types. Ade T, Zaucke F, Krug HF. The structure of organometals determines cytotoxicity and alteration of calcium homeostasis in HL-60 cells. Anal Bioanal Chem. 1996 Mar;354(5-6):609-14.

Development of a Ceramic Port Catheter with Antimicrobial Activity and Improved Fluid Dynamical Properties

Guggenbichler M. Guggenbichler, J.P. Univ. Erlangen/Nürnberg

Loschgestr. 15 D – 91054 Erlangen

Implantable port catheter systems assume increasing importance in clinical medicine. They contribute to safety and reliability of infusion therapy in critically and chronically ill patients and provide a number of therapeutic advantages in tumor therapy. Their use however is not without complications (e.g. infection and obstruction by thrombi)1

,2. Disadvantages also arise

from interference with tomographic imaging, chip formation and mechanical failure. For a number of reasons the materials used so far have not been completely satisfactory. Ceramic is virtually free of all these disadvantages. A new ceramic port system was developed utilizing the excellent biocompatibility. In addition antimicrobial activity and optimized fluid mechanical properties were added to this new device3.

An antimicrobial active ceramic material was developed by doting the base material with billions of Ag0 nano particles. These nano particles were added to traditional ceramic slurries of Titanium- and Aluminum oxide. Process optimization was performed varying the silver concentration and sinter temperatures. The concentration and shape of the silver particles in the ceramic material prior to and after the sintering process were investigated by electron microscopy and XPS. The antimicrobial activity was tested by the “Flask-Shake” method and the “Roll-On-Culture” method using Pseudomonas aeruginosa and Staphylococcus aureus as test organisms. The test results revealed an excellent antimicrobial activity at 2 % Ag concentration. There was a direct correlation between antimicrobial activity and sinter temperature. Cytotoxicity and biocompatibility were investigated and revealed also favorable results.

In addition the geometry of the construction was adapted to improve the fluid dynamics in the port chamber to avoid the formation of blood clots. The design was calculated and optimized with the FEM-method. The load presumptions for the calculation were 1 x 106 Pa inside pressure in the chamber in combination with a force of 100 N (insertion of the injection needle). The shape as well as the height of the component was optimized compared to existing products. A prototype was produced in slurry casting for verification of the experimental data.

This research project is close to clinical application and could provide a substantial improvement to patient care by prevention of complications and hospital admissions. 1 HARTKAMP A., VAN BOXTEL A., ZONNENBERG B., WITTEVEEN P.:

Totally Implantable Venous Access Devices: Evaluation of Complications and a Prospective Comparative Study of two Different Port Systems Neth. J. Med. 2000; 57: 215-223

2 WILDHABER B., KISTLER W., CATFISCH U.: Erfahrungen mit dem Port-A-.Cath.System bei Kindern Schweiz. Med. Wochenschr. 2000; 130: 732-738

3 GUGGENBICHLER M.: Entwicklung eines keramischen Port-Katheters mit optimierter Verwirbelung von Medikamenten, Blut- und Blutprodukten in der Portkammer sowie antiinfektiösen und antithrombotischen Eigenschaften Diplomarbeit, Lhst f. Feingerätebau, TU-München, 2004


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Establishment and validation of a biocompatibility assay for magnetic nanoparticles Frank Hauschild, Manuela Schwalbe, Norbert Buske1, Armin R. Kautz2, Cornelia Jörke, Klaus Höffken, Katharina Pachmann, Joachim H. Clement

Department of Internal Medicine II (Oncology, Hematology), Friedrich Schiller University Jena, Erlanger Allee 101, D-07740 Jena, 1BerlinHeart AG, Wiesenweg 10, D-12247 Berlin,

2Innovent e.V. Prüssingstr. 15, D-07745 Jena, Germany Introduction: Magnetic nanoparticles play an important role in purification of biomolecules, e.g. DNA, RNA, proteins, or specific cell types. There is growing evidence, that magnetic nanoparticles might be an important tool for transportation and localization of biomolecules or therapeutic agents in organisms. These future approaches make accurate testing of these particles necessary, especially with regard to their stability and biocompatibility. Therefore, we established a scheme for evaluation of magnetic nanoparticles. Methods: The breast cancer cell line MCF-7 and the lung cancer cell line A-549 were cultivated with DMEM + 10% fetal calf serum. The cells were incubated with various amounts of magnetic core/carboxymethyl-dextran nanoparticles with an average magnetite/maghemite core TEM-size varying between 3 and 15 nm (supplied by N.B. and A.R.K.). Magnetically labeled cells were separated by MACS. The separated cells were counted with a Coulter Counter Z2 and further analysed by FACS and RT-PCR. Results: The adherent monolayer cell lines MCF-7 and A-549 were incubated with carboxymethyl-dextran coated magnetic nanoparticles for 12 minutes. We added 0.5 – 4.0 µl of nanoparticle suspension/ml culture medium, because physical parameters varied within a broad range and were not useful for standardization. Cells were detached from the plastic surface and separated by MACS. Accutase, a novel detachment agent preserved the cell surface structures better than trypsin/EDTA, which might be the reason for higher yields of magnetically labeled cells. Propidium iodine (PI) staining revealed that almost all cells in the positive aqnd negative fraction were viable. A-549 cells showed a more rapide labeling and uptake of magnetic nanoparticles in comparison to MCF-7. This effect was independent from the sort of nanoparticles used. In order to analyse alterations of the incubated cells, we recultivate the cell suspensions for 24 hours and estimated the rate of apoptosis/necrosis with PI and Annexin V staining. Furthermore, we determined the expression of several genes involved in signal transduction (BMP-2; BMP receptor IA, BMP receptor IB, BMP receptor II) cell cycle control (ID1, ID2, ID3)and extracellular protease activity (PAI-2, Cathepsin H) with RT-PCR. The expression of all genes was unaltered with respect to the various incubation conditions, indicating that our magnetic nanoparticles do not affect important features of cell metabolism. Conclusion: We established a cell culture-based assay for testing the biocompatibility of magnetic nanoparticles. The cell cultures should be grown under defined conditions. The incubated cells should be recultvated after magnetic labeling and separation for 24 hours and then analysed morphologically and for apoptosis by FACS. This work was supported by the DFG-Schwerpunktprogramm SPP1104, grant CL 202/1-1


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Influence of surface roughness and biofunctionalisation on the cell behaviour of implant material

B. Hoffmann, R. Detsch, G. Ziegler

Friedrich-Baur-Research Institute for Biomaterials, University of Bayreuth, 95440 Bayreuth, Germany e-mail: [email protected]

Introduction The biological response of organisms to an implant can be caused by many different reasons, e.g. topography, chemical behaviour or porosity. There are many studies in literature which deal with this subject. In this study we want to combine two aspects. One aspect is the influence of topography, another aspect is the biofunctionalisation of these substrates with peptides. Sequences of amino acid like Arg-Gly-Asp-Ser (RGDS) are known to interact with integrin receptor located on the cell surface.

Materials and Methods TiAl6V4 discs (cleaned by sonification in a tenside solution, water and acetone) were used as substrates. Different roughness of the substrate was produced by mechanical treatment, and characterized by Ra-values and SEM.

The peptide sequences selected for biofunctionalisation were Arg-Gly-Asp-Ser (RGDS). As a negative control the peptide sequence Arg-Phe-Asp-Ser (RFDS) was chosen. In a first step the metallic (uncoated TiAl6V4 substrate) or ceramic surface (titanium oxide coated substrate) was activated with 3-aminopropyltriethoxysilane. In the following step 4-azido-1-fluoro-2-nitrobenzene was used as spacer. The peptides were linked via UV-light activation of the azido groups.

The mouse osteoblast-like-cells MC3T3-E1 were used for the in vitro cell investigations. The samples were washed with PBS and the surface attached cells were trypsinised after an incubation time of 48 hours. The cell proliferation was determined either by counting the trypsinised cells with a cell counter or by measuring the integration of BrdU in the synthesized DNA. The cell viability was analyzed by the WST-1 test (Roche, Germany). Results Varying roughness of the substrate (TiAl6V4) was selected to indicate the effect of topography. One experiment, which deals with sand blasted (Ra = 0.99 µm), untreated (Ra = 0.37 µm) and polished substrates (Ra = 0.023 µm) analyses great differences in roughness. In a second experiment the influence of a smaller variation in roughness was studied by preparing different grinding steps (Ra-values vary from 0.059 µm to 0.118 µm). By dip coating and subsequent calcination at 500 °C dense coatings of titanium were achieved on the substrates (TiAl6V4) with different roughness. The thickness of the layers totals almost 140 nm. Additionally to this inorganic modification, the surface was modified by covalently immobilized peptides.


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The chosen amino-sequence RGD is known to act with integrin receptors of the cells. Peptides were immobilized on amino-functionalised substrates by using 4-azido-1-fluoro-2-nitrobenzene as spacer.

Fig 1.: Cell behaviour on sand blasted TiAl6V4 in comparison with different surface modifications.

Fig 2.: Cell behaviour on polished TiAl6V4 in comparison with different surface modifications.

In vitro investigations of the surfaces indicate an increase of cell number and vitality with decreasing roughness (figs 1 and 2). The polished surface shows good cell behaviour compared to the rough surfaces. The results of the sand blasted surfaces can be improved by RGD-functionalisation, so that the cell behaviour shows similar results as the polished surfaces. Interestingly, the polished surfaces cannot further be improved by RGD functionalisation. These results show, that topography has a more significant influence than the functionalisation with RGD-peptides.

Acknowledgements This work was sponsored by the DFG (Schwerpunktprogramm “Grenzfläche zwischen Werkstoff und Biosystem” (SSP 1100)).

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THE EFFECT OF SIMVASTATIN ON POLYETHYLENE PARTICLE-INDUCED OSTEOLYSIS

*+von Knoch F; **Heckelei A; **Wedemeyer C; **Saxler G; ***Hilken G; ****Henschke F; **Löer F; **von Knoch M

+*Department of Orthopaedic Surgery, Kantonspital Chur, Chur, Switzerland. 617-724-1923, [email protected]

Introduction Periprosthetic particle-induced osteolysis with subsequent aseptic loosening remains the most common and important long-term complication associated with total hip replacement. In the face of these challenges to total hip arthroplasty, noninvasive treatment alternatives are becoming increasingly desirable both for prevention and treatment of periprothetic osteolysis. Bisphosphonates inhibit osteoclastic bone resorption by interfering with the mevalonate pathway. The effectiveness of these drugs in preventing experimental wear debris-induced bone loss has been demonstrated in a canine model by Shanbhag et al. (1) as well as in a murine model by our group (2). Statins, a class of drugs clinically used to suppress hepatic cholesterol synthesis, act also on the mevalonate pathway by inhibiting HMG-CoA reductase. These drugs have been proven to suppress osteoclastic bone resorption both in-vitro and in-vivo (3,4). Thus we hypothesized that statins, similar to bisphosphonates, could suppress particle-induced osteolysis through effects on osteoclastic bone resorption. This study investigated the effects of simvastatin on ultra-high molecular weight polyethylene (UHMWPE) particle-induced osteolysis in a murine calvarial model.

Materials and Methods Animal model: Recently we introduced a murine calvarial model of UHMWPE particle-induced osteolysis (5), serving as an extension of the well-established titanium based model (6). Our model was applied in twenty-one C57BL/J6 mice in accordance with the institutional animal use committee approval. Animals were equally randomized to three groups. Group I animals underwent sham surgery only, group II received UHMWPE particles (about 6 x 106 particles per

animal) and group III received particles and simvastatin (Zocor, Merck, Rahway, NJ) at a daily dose of ∼ 120mg/kg from day 0 until sacrifice, supplemented to a professional diet. One mouse from group I did not recover from anesthesia and was excluded. Retrieval and processing of specimen: Calvaria were harvested 14 days postop and prepared using techniques of undecalcified hard tissue processing. Sections were histochemically stained with Giemsa as well as for tartrate-specific acid phosphatase (TRAP). Bone histomorphometry: Bone resorption was measured as resorption within the midline suture using Giemsa staining as described previously (6). Osteoclast numbers were determined per high power field using TRAP-staining.

Statistical analysis was performed using a one-way ANOVA and a post-hoc Student’s two tailed t-test. All p-values were compared to an α-value of 0.05 to determine significance.

Results Histologically, there was formation of fibrous and granulomatous scar tissue overlying the calvarium (Fig. 1A-C). UHMWPE particle implantation without any further intervention (group II) induced pronounced bone resorption compared to sham controls (group I). Treatment with simvastatin (group III) significantly diminished these UHMWPE particle-mediated effects. Bone resorption in midline suture was 0.094 ± 0.007 mm2 in group I, 0.25 ± 0.025 mm2 in group II, and 0.131 ± 0.02 mm2 in group II (Fig. 2). There was a statistical significant difference in bone resorption between group I and II (p= 0.018), group II and III (p= 0.002) (ANOVA p=0.00003). Osteoclast numbers were 15.3 ± 3.6 in group I, 48.7 ± 7.1 in group II and 6.2 ±


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3.1 in group III (Fig. 3). There was a statistical significant difference in osteoclast numbers between Group I and II (p=0.0085) and Group II and III (p=0.00022) (ANOVA p=0.00002).

Discussion This study demonstrates that simvastatin markedly diminished UHMWPE particle-induced osteolysis in a murine calvarial model. Our results indicate that these beneficial effects primarily rely on the inhibition of osteoclast formation and osteoclast function, although the main action of statins drugs on bone turnover is believed to be the stimulation of osteoblastic bone formation (3). Clinical data as how statins affect wear particle-induced osteolysis around total joint replacement in humans is not available to date. Overall, our new finding points to the possibility that statins like simvastatin may be

potential candidates for noninvasive prevention and treatment of periprosthetic osteolysis after total joint replacement. An increasing understanding of the complex interactions of statins with bone metabolism might open up a broad application of these drugs to critically improve the durability of implants in orthopaedic surgery.

Figure 1: Typical calvarial histology (original x10): (A) control SHAM mice (group I), (B) after UHMWPE particle treatment (group II), and (C) after particles and simvastatin treatment (group III).

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References 1. Shanbhag AS et al. Clin Orthop 344:33-43, 1997. 2. von Knoch M et al. Biomaterials 2004 in press 3. Mundy G et al. Science 286:1946-1949, 1999. 4. Staal et al. J Bone Miner Res 18:88-96, 2003. 5. von Knoch M et al. J Orthop Res 22:237-43, 2004. 6. Schwarz E et al. J Orthop Res 18:472-80, 2000.

Acknowledgement: Supported by the German Research Foundation (DFG) Bonn, Germany (Grant KN 553/2).

** Dpt. of Orthopaedics, University of Duisburg-Essen, Essen, Germany ***Central Animal Laboratory, University of Duisburg-Essen, Germany ****Patholog. Institut am Johannisstift, Paderborn, Germany


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Tuberculostatic Hydrazides of Isonicotinic Acid:

Isoniazide, Propioazide, Ftivazidum and Flurenizide. Quantum Mechanical Simulation.

N.Kozak, Yu.Nizelskii

Institute of Macromolecular Chemistry NAS of Ukraine, Kyiv, e-mail: [email protected]

Isonicotinic hydrazide (isoniazide) and its derivatives are prevalent inhibitors in current

treatments for tubercular infections. Although isoniazide is applied since 1952 the mechanism of its tuberculostatic action is opaque. Analysis of interactions of drug molecule with biomolecules is of importance for medicine and clinical pharmacology. Data concerning the electron structure of hydrazides of isonicotinic acid derivatives are absent in literature.

Using semiempirical MINDO/3, PM3 and ab initio with extended basis set quantum chemical methods the calculations were performed of conformations and electron structure of isonicotinic hydrazide (I isoniazide), isopropyl-1-isonicotinic hydrazide (II propioazide), N-[3-methoxy-4-oxybenzylidene]-isonicotinic hydrazide (III ftivazidum) and N-[9-fluorene]-isonicotinic hydrazide (IV flurenizide) both in ketonic- and enol form.

I II III

IV

In above series compound IV contains additional important pharmacophore - fluorene

as compared with I and possesses practically identical tuberculostatic activity. Toxicity of IV being essentially lower.

Optimized geometry parameters, polarizability, dipole moments and molar volume of tautomers I-IV were analyzed as well as their molecular electrostatic potential (MESP). The last can be used to predict possible channels of positive charged particle attack as the initial stage of complex formation.

The structure of complexes of IV with Li, K, Na, Ca(2+), Cu(2+) ion were optimized. The peculiarities of chelate environment of Ca(2+), Cu(2+) in the complexes is discussed.

The correlation structure – properties – pharmacological activity to be revealed in the future can help in selective design and preclinical testing of antitubercular drugs.


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Reaction Analysis of Chromosulsuric Acid Action on Transition Metals at High Temperatures

D. Lattner and H.P. Jennissen Universität Duisburg-Essen, Universitätsklinikum, Essen, Institut für Physiologische Chemie

Biocoating or biofunctionalization of metallic implant materials to enhance their tissue compatibility has become an expanding field in biomaterial research. We have established a methodology for the biocoating of bone implantable metals such as titanium with morphogens or growth factors such as bone morphogenic protein 2 (BMP-2) [1-5]. A prerequisite for these procedures is the creation of an ultra-hydrophilic layer on the respective metal surfaces, which is established by an initial treatment with chromosulfuric acid (CSA) [1]. Several years ago we discovered that transition metals (Ti, CoCr, steel) display ultra- hydrophilic behaviour after treatment with chromosulfuric acid at elevated temperatures (> 200 °C). According to current definition the hydrophilicity of a material is regarded as the equivalent to its free surface energy. High surface energies between a solid and a liquid (aqueous) phase originate in weak non-covalent interactions such as van-der-Waals interactions or hydrogen bonding, which enhance the tendency for wetting. In general, we consider a material ultra-hydrophilic, when its dynamic contact angle (Wilhemy plate method) is in the region between 0° – 10° in addition to ~ 0° hysteresis [3]. To investigate the physicochemical processes involved in chromosulfuric acid etching, electropolished and anodically oxidized titanium miniplates (size: 1.0 x 10 x 15 mm) were employed as model materials. Fundamental process parameters such as temperature and duration of CSA treatment were correlated with the physical properties (dynamic contact angle, surface area increase) as well as the chemical composition of the metal surface layer. All materials under investigation displayed ultra-hydrophilic behaviour after etching. Current results of dynamic contact angle measurements indicate that CSA etching is a multiple stage process, in which the sample hydrophilicity goes through a minimum with increasing duration of exposure to chromosulforic acid. On the other hand roughness measurements via confocal laser scanning microscope (CLSM) display a basically time-independent behaviour for all samples after 10 minutes of exposure. These results suggest that the ultra-hydrophilicity of the enhanced metal surfaces is mainly governed by their surface chemistry and to a lesser degree by surface microgeometry (roughness). [1] Jennissen H.P., Zumbrink T., Chatzinikolaidou M., Steppuhn J.: Biocoating of Implants with Mediator

Molecules : Surface Enhancement of Metals by Treatment with Chromosulfuric Acid, Mat.-wiss. u. Werkstofftechn. 30 (1999), 838-845

[2] Voggenreiter G., Hartl K., Assenmacher S., Chatzinikolaidou M., Rumpf H.M., Jennissen H.P.: Assesment of Biological Activity of Chemically Immobilized rhBMP-2 on Titanium Surfaces in vivo, Mat.-wiss. u. Werkstofftechn. 32 (2001), 942-948

[3] Jennissen H.P.: Ultra-hydrophile metallische Biomaterialien, Biomaterialien 2 1 (2001), 45-53 [4] Chatzinikolaidou M., Laub M., Rumpf H., Jennissen H.P.: Biocoating of Electropolished and Ultra-

hydrophilic Titanium and Cobalt Chromium Molybdenum Alloy Surfaces with Proteins, Mat.-wiss. u. Werkstofftechn. 33 (2002), 720-727

[5] Lichtinger T.K., Müller R.T., Schürmann N., Oldenburg M., Wiemann M., Chatzinikolaidou M.,

Rumpf H.M., Jennissen H.P.: Osseointegration of Titanium Implants by Addition of Recombinant Bone Morphogenic Protein 2 (rhBMP-2), Mat.-wiss. u. Werkstofftechn. 32 (2001), 937-941


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Tissue deformation modelling: measurements and simulation

H.-G. Lipinski, R. Mahnke, N. Schwichler, A. Akbar-Rabii

University of Applied Science (FH) Dortmund; Dept. of Medical Informatics, Emil-Figge-Str. 42, D-44227 Dortmund; email to [email protected]

Mechanical tissue deformation can be completely described by the basic elasto-mechanical equation which contains a system of partial differential equations. A discrete solution of these differential equations can be found by forming a finite element model (FEM) of the process. A FEM, however, requires a great number of parameters including their numerical values. These values are often unknown or could not simply be measured by experiments. Consequently, models of tissue deformation, necessary e.g. for OP simulators, which are based on physical parameters are rather complicated and probably useless for real time simulators. Therefore, we developed a graphical method to simulate a simple kind of tissue deformation. First, we measured a real tissue deformation. A tissue membrane (ca. 5x5 cm square) was put into an experimental chamber which allows for a simple deformation of the tissue. The deformation was performed by a small rod forming a “tissue dent”. The maximum height of the “dent” was approximately 3 cm. The dome of the “dent” ended in a point if the tissue was thin (membrane) and the deformation was performed by a thin rod. Otherwise, the dome was rounded, if the tissue thickness and/or the rod thickness was increased. After the tissue deformation was performed we measured the coordinates of approximately 100 discrete surface elements of the “dent” by the aid of a conventional surface 3D-scanning apparatus (accuracy < 1 mm). The data of the surface coordinates were stored into a data base for further analysis.

Second, we developed a mathematical model of the deformation. The deformation can be described by a continuous 2D-function z(x,y) with has only one maximum. We scaled the function exponentially by z=z(ζp,ξq), which allows to simulate different dome types of the generated dent by changing the numerical values of the parameters p and q (p and q are real positive numbers; p and q are equal if the deformation is completely symmetrical). We selected a cosine-function (separable into two basic functions f(x) und g(y)) to realize the model:

)]ycos(1[)]xcos(1[4z

)y(g)x(fz)y,x(z qp00 ⋅π+⋅⋅π+⋅=⋅⋅=

( | x | < 1; | y | < 1 ) The maximum of the dent, z0, is located at x=y=0.

Third, we fitted model data and surface data collected by the 3D-scanner apparatus. We found that the model z(x,y) fitted well real deformation data at various dome types generated by different tissue thickness or rod diameters, respectively.

Finally, we used the model to perform a virtual tissue deformation. A virtual membrane, represented by the model z(x,y) was deformed by a virtual rod. For this purpose we used Sensable®’s Phantom stick. This manipulator allows to perform a virtual deformation including an haptical 3D-feedback. The tip of the virtual rod could be moved from z0=0 up to a defined maximum height z0=z0

max. Using this computerized “virtual reality”-technique we can completely generate a simple virtual tissue deformation according to the model z(x,y) described above.


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A computerized method to analyse bone cell mobility in vitro

S. Magosch+), H.-G. Lipinski+), L. Winkler*), M. Wiemann*), D. Bingmann*)

+) University of Applied Science (FH) Dortmund; Dept. of Medical Informatics, Emil-Figge-Str. 42, D-44227 Dortmund; email to [email protected]

*) University Duisburg-Essen, Physiological Institute, Hufelandstr. 55, 45147 Essen We developed a computer program (C++ program, running on a standard PC) which allows to analyse patterns of bone cell mobility in vitro. Vital bone cells (cell culture; 500 < N < 1200; MC3 T3-E1) were prepared and continuously scanned by a digital camera at different times (an image stack of 400 pictures with a resolution of 1232 x 972 pixels which were scanned during several hours). After a pre-processing (including repairing of artefacts and noise minimizing) the digital grey tone images were converted into binary images by a standard grey tone thresholding method. From binary images the cell volume could simply be calculated. Next, the contour of each cell was detected by the well known Marr-Hildreth filtering technique. The contour detection was completed by shrinking the contour up to one pixel thickness using the Zhang-Suen skeletonization algorithm. The contour length of each cell (“circumference”) could simply be calculated by counting the number of the contour pixels. The mean values of both coordinates of the contour pixels allows to compute additionally the cell centre of mass (com).

The cell movement was detected by measuring the distance between the com-coordinates of a cell observed at two different times. From the distance value a vector array of cell velocity derived which could be separated into two components: the “direction” of the vector and its “amount”. The amount value describes the velocity of a cell, the direction value contains the direction in which the cell actually moves. The movement of the complete number of cells in the image stack could be visualized by the aid of a global velocity vector array. Hence, any cell of the image stack could be characterized by (i) its actual position, (ii) its actual velocity, and (iii) its actual circumference/volume.

The program allows to follow up the movement of the each cell. We can calculate the complete distance moved by a cell, its actual volume and volume variations as a function of time. Furthermore, the velocity of each cell and the direction of its movement as well as the distance between the coordinates at the beginning and at the end of the observation. Finally, the movement process could be presented as an animation. The programs runs fast on standard PCs (70 pictures per minute can by analysed if an Intel processor 2 GHz is used). All generated data can be stored in a separate data base connectable to external data analysis programs like Excel.


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Deposition of calcium-phosphate layers on the materials of medical implants by magnetron sputtering

V.F. Pichugin, A.A. Bulycheva, S.I. Tverdokhlebov, R.A. Surmenev, I.A. Khlusov*,

V.V. Sohoreva**, T.G. Volova***, E.I. Shishatskaya *** *Polytechnic University, 30 Lenin Av., Tomsk, Russia, e-mail: [email protected]

*Siberian State Medical University, Tomsk, Russia **Nuclear Physics Institute, 2a Lenin Av., Tomsk, Russia

***Biophysics Institute, 660036, Akademgorodok, Krasnoyarsk, Russia

Formation of biocompatible coating on the materials of medical implants such as titanium, chrome-nickel alloys, bioceramics and polymers (in particular biodegradable polymers of bioorganic origin such as polyoxibutyrate) is actual problem.

The aim of this work is application of the magnetron sputtering process to the deposition of calcium-phosphate (Ca-P) coating on the materials of medical implants.

The installation for complex ion – plasma surface treatment of different kind of materials was made. Installation consists of vacuum chamber with ion accelerator up to 10 keV and magnetron spattering system. Matrix cathode of magnetron system is multi-component and contains 50% of natural hydroxyapatite and 50% zirconium oxide. Hydroxyapatite contains 35% Ca & P (Ca/P ratio is 1.67) and 60% oxygen. The rest 5% is impurity. Ca-P thin films were deposited by DC reactive magnetron spattering with Ar ion-beam assistance. Chemical composition, mechanical and structural properties of thin films was investigated by the methods of Rutherford Backscattering of α-particles, SIMS technique, Electron Auger Spectroscopy. Thickness of investigated films was no more than 1 µm. The quantity of normal peeling of coating from substrate was at least 40 MPa. Microhardness of Ca-P coating on titanium substratum is 10,900 MPa and on stainless steel is 10,440 Mpa one. The results of element analysis demonstrate the presence of P, Ti, Zr, Ca, Fe and O in Ca-P coating. Distribution of elements in depth of coating is inhomogeneous. The Ca/P ratio is about one-half and thin films have high packing density, good adhesion, and smooth surface.

Biological tests have been carried out for seven - days extract of samples obtained in isotonic saline in accordance with prescription of ISO 10993-5. Extracts were injected in liquid rat’s bone marrow cell culture in amount of ¼ from total volume (2 ml) of cellular suspension. Cytotoxicity test was done with trypane blue. Indices of conventional unsurfaced substrata extracts were used for control. pH of extract was determine also. Testing in vitro demonstrates satisfactory toxicological properties of obtained composite (metal + Ca-P) implants. Bone marrow cells survivability are 89.56 % (at 90.49 % in the control) for titanium substratum and 97 % for stainless steel one.

Testing in vivo show that implantation of obtained composites to three-month mice does not change their mass. Condition of thymus and spleen indicates the absence of immune reactions on implanted composites. Examination of tissue reaction shows that there is no inflammation and infectious development for all groups of observations. Cohesion of Ca-P coating to mucous tunic is maximal for sample of stainless steel and minimal for polyoxibutyrate one. Maximal biocompatibility of magnetron Ca-P coating is characteristic for stainless steel substrata. Probability of tissue lamina formation from column of bone marrow on such a film is small.

Thus, the biocompatibility of magnetron Ca-P films with tissue is high for all substrata and they do not provoke inflammatory as well as immune and toxic reactions. This work is supported by the Russian Foundation of Basic Research, grant 02-02-16280.


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An improved 96-well microplate assay to analyze osteogenic activity of bone morphogenetic proteins

Marko Pietraszczyk, Peter Hortschansky, Klaus Höffken, Stefan Wölfl, Joachim H.

Clement Department of Internal Medicine II and Branch Molecular Biology, Friedrich Schiller

University Jena, Erlanger Allee 101, D-07740 Jena, Department for Applied Microbiology, Hans-Knoell-Institute for Natural Product Research, Beutenbergstrasse 11, D-07745 Jena,

Germany Bone morphogenetic proteins (BMPs) play a vital role in the regulation of cell metabolism. A couple of BMPs became available as recombinant proteins synthesized in vitro in different expression systems. To ensure that these proteins are fully functional a proof of biological activity of these proteins is required. The ability of the promyoblast cell line C2C12 to

differentiate into osteoblast-like cells after application of inducing factors is an established method for this purpose. To make this assay truly reliable various parameters should be monitored, leading to a large number of samples, which must be analyzed. To reduce experimental variability and time-consuming re-evaluations, we optimized the cell culture regime and develop a technical protocol to measure BMP activity in a 96-well microplate assay.

The C2C12 cell line was grown in Dulbecco´s modified Eagle´s medium (DMEM) supplemented with 2 % FCS in an incubator at 5 % CO2 , 37 °C and 90 % humid atmosphere. Recombinant human BMP-2, BMP-6, BMP-7 and Noggin were produced in E. coli as MalE fusion proteins and reconstituted resulting in homodimeric active molecules and in case of BMP-2/6 as heteromer. The osteogenic activity of bone morphogenetic proteins was measured with a colorimetric assay (ALP-SIGMA, Sigma, Deisenhofen, Germany).

We established a reliable test taking several critical parameters into account, especially culture conditions and growth behavior of

the cells. Because C2C12 cells differentiate spontaneously when confluent a strict time-

schedule for seeding and cultivation is necessary to exclude confluence (Fig. 1). A key role plays the accurate long-term cultivation of the C2C12 cells. Additionally, the incubation and the handling of the cell cultures and the preparation and measurement of the samples should

be reproducible. All these parameters can be fulfilled with our 96-well microplate assay. Moreover, this assay allows us to process a large number of samples under identical

conditions, making the results even more accessible for direct comparison. We think that this improved 96-well microplate assay will provide a good tool for comparing and

standardization of biological activitys of recombinant BMPs.

t0 = 0 h x0 = 1,50x104

∆tpi = 3 hti = 3 h xi = 1,60x104 (calculated)∆ti = 48 h te = 51 h xe = 4,45x104 (calculated)

Figure 1: Time schedule of the 96-well microplate assay based on C2C12 cells


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Coating of nickel-titanium shape-memory alloys with calcium phosphate by plasma-spraying

O. Prymak1 D. Bogdanski,2 M. Köller,2 S.A. Esenwein,2

G. Muhr,2 M. Epple*,1 1 Institute of Inorganic Chemistry, University of Duisburg-Essen, Campus Essen,

Universitaetsstrasse 5-7, D-45117 Essen, Germany 2 Department of Surgery, BG Kliniken Bergmannsheil – Universitätsklinik, Bürkle-de-la-

Camp-Platz 1, D-44780 Bochum, Germany, University of Bochum, Universitaetsstrasse 150, D-44780 Bochum, Germany

Plates of superelastic nickel-titanium shape memory alloy (NiTi) were coated with calcium phosphate (hydroxyapatite) by high-temperature plasma-spraying. The porous layer of about 100 µm thickness showed a good adhesion to the metallic substrate that withstood bending of the plate but detached upon cutting the plate. The biocompatibility was tested by cultivation of blood cells (whole blood and isolated granulocytes [a subpopulation of blood leukocytes]). As substrates, pure NiTi, plasma-spray-coated NiTi and calcium phosphate-coated NiTi prepared by a dip-coating process were used. The adhesion of whole blood cells to all materials was not significantly different. In contrast, isolated granulocytes showed an increased adhesion to both calcium phosphate-coated NiTi samples. However, compared to non-coated NiTi or dip-coated NiTi, the number of dead granulocytes adherent to plasma-sprayed surfaces was significantly increased for isolated granulocytes (p<0.01). We gratefully acknowledge financial support by the Sonderforschungsbereich 459, Bochum.


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Fatigue of orthodontic Nickel-Titanium (NiTi) wires in different fluids under constant mechanical stress

O. Prymak,1 A. Klocke,2 B. Kahl-Nieke,2 M. Epple*,1


Universitaetsstrasse 5-7, D-45117 Essen, Germany 2 Department of Orthodontics, University Hospital Hamburg-Eppendorf, Martinistr. 52, D-

20246 Hamburg, Germany The aim of this study was to analyze in-vitro the fatigue resistance of NiTi and CuNiTi orthodontic wires when subjected to forces and fluids which are present intraorally. The wires were subjected to dynamic mechanical analysis (DMA) while they were immersed into different fluids with mechanical loading parameters similar to those they are subjected to in the mouth. The characteristic temperatures of transitions and a rough surface structure on the perimeter of the wires were determined by differential scanning calorimetry (DSC) and scanning electron microscopy (SEM), respectively, before and after the DMA experiments. Stainless steel wires were used for comparison. In general, NiTi wires fractured earlier than the stainless steel specimens. Survival times were lower for the NiTi wires when immersed in fluids (water, citric acid, NaCl solution, artificial saliva, fluoridated artificial saliva) than in air. SEM surface analysis showed that the NiTi and CuNiTi wires had a rougher surface than steel wires. The fracture occurred within a short number of loading cycles. Until fracture, the mechanical properties remained mostly constant. Particular emphasis was laid on fracture properties of surface-nitridated NiTi wires. We gratefully acknowledge financial support by the Sonderforschungsbereich 459, Bochum.


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Correlation of Structural Properties of Commercial DLC Coatings to their Tribological Performance in Biomedicinical

Applications

S. Reuter1, B. Weßkamp2, R. Büscher3, A. Fischer3, B. Barden2, F. Löer2, V. Buck1*

1Thin film technology group FB 7,2Department of Orthopaedic Surgery,3Materials Technology IPE University of Duisburg Essen, Germany

[email protected] In medicine, artificial joints have been used very successfully for many years now. Worldwide about 600.000 knee- and hip-endoprostheses are implanted per year. But many open questions in basic research remain. Especially the stability time of a prosthesis, averaging from 10 to 15 years, is unsatisfying. The main cause for failure is the loosening of the implant due to osteolysis, which is caused by wear particles. In recent times prostheses were coated with amorphous carbon (a-C; a-C:H), to avoid the development of wear particles, because a-C and a-C:H coatings are characterised by excellent biocompatibility, low friction and low wear. To optimize the coatings for this specific application, 5 commercial coatings, differing in sp3/sp2 ratio, H-content, interface design etc., on P2000 steel (plus TiN, and CrN) are compared under conditions derived from applications in human prostheses using disc on pin test. The results are discussed, showing differences in the tribological behaviour, confirming that the expression “diamond like carbon” is not sufficient to characterize hard carbon coatings. Correlations between structural properties and tribological performance are given. Keywords: DLC, Body implants, Tripological performance, Raman-Spectroscopy


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of 167 Pages

Sharpening and de-noising algorithms to enhance the quality of digital confocal colour images of cultured vital bone cells

A. Roth+), H.-G. Lipinski+), M. Wiemann*), D. Bingmann*)

+) University of Applied Science (FH) Dortmund; Dept. of Medical Informatics, Emil-Figge-Str. 42, D-44227 Dortmund; email to [email protected]

*) University of Duisburg-Essen, Physiological Institute, Hufelandstr. 55, 45147 Essen Digital confocal colour images gathered from vital bone cells are often superposed by high frequency noise signals. The standard median algorithm is commonly used to eliminate such noise signals from the digital images. But there are some leaks in this algorithm which can significantly decrease the quality of the filtered image. On the one hand the median filter eliminates noise signal by low pass filtering. On the other hand, however, the image contrast is reduced and detailed contours of morphological structures may also be eliminated by that algorithm. To avoid these effects we first developed a modified median filtering algorithm. A 3x3 pixel template was shifted over the image matrix. At a fixed position a square of 9 pixels of the image are covered by the template (so called “template covered image matrix area, TCIA”). Using the standard median algorithm the RGB-values of the central pixel (CP) of the TCIA is simply replaced by the median RGB-value of the 9 pixels. Our algorithm, however, compares the computed median value with the original CP data. If the difference between the CP value and the median value is less than a certain value (which has to be explored empirically) the CP value is replaced by the median value. Otherwise, the pixel value will not be changed. Next, the template is shifted and the procedure starts again. Shifting of the template takes place until the complete image is computed. This simple algorithm runs on standard PCs as a C++ program and allows to reduce the noise level efficiently without image contrast decreasing. A 512x512 image matrix filtering requires less than 1 s. Next we developed an algorithm which allows to enhance the contrast of non-disturbed digital colour images of vital bone cells. Conventional contrast enhancing (so called “sharpening”) algorithms increase object contours but often decrease the total quality of the images. Our algorithm, which is based on a modification of the Fourier spectrum of the image, avoids this side effect. The complex spectral data are weighted by a non-linear function, which could be expressed by only one parameter. The degree of the contrast enhancement can be adjusted by this parameter. Hence, the algorithm requires first a fast Fourier transform (FFT) of the image matrix. Second, the user has to select a certain parameter value. Third, the spectral data are modified by the weight function. Forth, the modified spectral data are re-transferred by inverse FFT. Finally, the modified image is presented on the screen. We can show that the contrast of the image data can be efficiently enhanced by this method. Consequently, fine detailed morphological structures can be clearly visualized. A C++-program was developed which is based on this algorithm. It runs fast on standard PCs. The contrast enhancement of a 512x512 image matrix requires less than 1 s per color (RGB) channel.


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LOCALIZATION OF SP- AND CGRP-IMMUNOPOSITIVE NERVE FIBRES AND OF THE NK1-RECEPTOR IN THE HIP JOINT OF PATIENTS WITH FEMORAL

NECK FRACTURE, OSTEOARTHRITIS, AND FAILED HIP IMPLANTS

G. Saxler1 F. Löer1 M. Skumavc1 U. Hanesch2

1 Department of Orthopaedic Surgery, University of Duisburg-Essen, Hufelandstr. 55, D-45122 Essen 2 Department of Physiology, University of Wuerzburg, Roentgenring 9, D-

97070 Wuerzburg, Corresponding author: Guido Saxler, MD, Department of Orthopaedic Surgery, University of Duisburg-Essen, Hufelandstr. 55, D-45122 Essen, Tel.: +49 201 4089 2139, FAX: +49 201

4089 2722, Email: [email protected] Introduction: Recent studies on osteoarthritis have focused on nociceptive substance-P (SP) and calcitonin gene-related peptide (CGRP) containing afferent nerve fibres. These neuropeptides are modulators of nociceptive sensation and they are involved in neurogenic joint inflammation when released from the primary afferents upon nociceptive stimulation. The effects of SP are known to be mainly mediated by the tachykinin receptor neurokinin 1 (NK1-R).

Aim: The purpose of this study was to investigate the innervation of SP- and CGRP- immunopositive nerve fibres and the distribution of the NK1-R in the hip joint of controls with femoral neck fracture (group 1), in patients with painful hip osteoarthritis (group 2) and in patients with failed hip implants who did not report pain (group 3).

Material and Methods: The hip joint capsule (groups 1-3), the soft tissue of the fossa acetabuli (group1), and the tissue of implant-bone interface (group 3) were resected, cut into small blocks of about 5x5 mm and immersion fixed in Zamboni's fixative. The specimen were frozen, cut in 50µm sections and immunostained using a standard immunofluoresence staining protocol.

Results: In the joint capsule of controls (group 1) and of patients with osteoarthritis (group 2) SP- and CGRP-immunoreactive (-ir) nerve fibres were mainly found in the subintimal part of the synovial layer (Fig. 1). In the capsule as well as in the soft tissue of the fossa acetabuli they were either grouped in small bundles or ran as single fibres through the tissue (Fig. 2). A greater portion accompanied small blood vessels. In group 1 and 2 CGRP-ir afferents were more abundant than SP-ir ones. In osteoarthritic joints (group 2) the number of immunopositive afferents increased about threefold. However, patients with failed hip arthroplasties lack these neuropeptide containing afferents.

In groups 1 and 2 the NK1 receptor could be localized in the wall of venous vessels, on Schwann cell of nerve bundles and on nerve fibres (Fig. 3). In group 2 NK1-R was additionally found on immune cells. Apart from that the staining pattern did not change in the osteoarthritis group but the number of stained receptor molecules was enhanced. In group 3 no NK1-R could be found.

Conclusion: This study clearly shows that peptidergic nociceptive afferent nerve fibres are present in the human hip joint capsule and the soft tissue of the fossa acetabuli under normal conditions, suggesting that pain may originate in these tissues. Indeed, in naturally occuring painful osteoarthritis in man the number of SP- and CGRP-positive nerve fibres was increased. This may be explained either by an upregulation of peptide synthesis in neurons which produce lower amounts of the peptides under normal conditions or by an recruitment of neurons for synthesis. In both cases, the signal is most likely arising from the arthritic joint. We, therefore, assume that the hip joint capsule and the soft tissue of the fossa acetabuli are important triggers of nociception. This is supported by the fact that patients with loosened total hip arthroplasties, where


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we failed to detect neuropeptide containing afferents, did not feel pain. The changes in the density of NK1-R immunostaining seen in this study fit well to the signalling of pain in patients with osteoarthritis resp. failed hip implants. The upregulation of NK1 receptor molecules in response to arthritic stages provides further evidence for a mechanism involving SP in the maintenance of a painful joint disease and in mediating noxious stimuli from the periphery.

Figures

Fig.1: CGRP immunopositive nerve fibres in the capsule of a patient with Osteoarthritis (arrows), nerve fibre ending (vertical arrow)

Fig.2: Small SP immunopositive peripheral nerve, branching off (superior arrow) into the adipose and collagen tissue, located close to a blood vessel in the soft tissue of the acetabular fossa

Fig.3: Granule-like NK1-R immunoreactivity on a small nerve in the adipose tissue of the acetabular fossa

50µm


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Influence of Condensation Technique on Quality of Direct Gold Restorations

Schmitz, I.*, Kamann, W.**, Gängler, P.** and K.M. Müller*

*German Mesothelioma Register and Institute of Pathology, BG- Clinic Bergmannsheil Bochum, University Clinic

** University of Witten-Herdecke, Dental Medicine Introduction There are only few studies available concerning the survival rate and qualitative parameters of direct gold restorations. Techniques and materials used are important parameters for survival rates of restorations.

Therefore, direct gold restorations in Black-Class I and V-cavities were compacted using the foil technique, the powdered gold technique and the mat cohesive technique and were followed up over a period of 12 years. Aim Aim of the study was to characterize and to compare the surface quality and marginal integrity of direct gold restorations condensed by different techniques. Methods In Black-Class-I cavities 6 direct gold restorations were condensed using the foil technique. Another 13 restorations were condensed according to the principles of the mat foil technique. 6 restorationsn were condensed with powdered gold only. In Black-Class V cavities 7 direct gold restorations were condensed using the the powdered gold technique and 16 using the mat cohesive technique.

After 12 years the direct gold restorations were rated clinically according to the C- criteria (CPM- Index). Additionally replicas were made with a dental impression material using a 2-stage replica technique and a low viscosity resin. The replicas were rated according to the M- criteria (CPM-Index). SEM invastigations were done with the DSM 940 (LEO/ZEISS).

Results In stress areas of Black-Class I-cavities the restorations condensed with gold foil and the mat cohesive technique had surface and marginal parameters superior to those condensed with powdered gold only. In Black-Class V- cavities the marginal adaption of mat cohesive restorations was superior to powdered gold restorations but the surface parameters were similar.

Conclusions The long term qualitative parameter of direct gold restorations are influenced by the condensation technique.

The indication of powdered gold restorations should remain restricted to non-stress areas.


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Transfection of cells with custom-made calcium phosphate nanoparticles

V. Sokolova,1 T. Welzel,1 W. Meyer-Zaika,1 I. Radtke,2 R. Heumann,2 M. Epple*,1


Universitaetsstrasse 5-7, D-45117 Essen, Germany 2 Chair of Biochemistry, Molecular Neurobiochemistry, University of Bochum,

Universitaetsstrasse 150, D-44780 Bochum, Germany Functionalised nanoparticles of calcium phosphate were prepared by controlled precipitation from aqueous solution, followed by coating with DNA. A successful transfection of transformed human endothelial cells was accomplished by adding the dispersion of nanoparticles to the cell culture. The functionalised nanoparticles form a stable colloid and did not lose their ability for cell transfection during 2-3 weeks of storage. The nanoparticles are approximately spherical with diameters of 10 to 20 nm and probably covered and stabilised by a hull of DNA. References 1. T. Welzel, I. Radtke, W. Meyer-Zaika, R. Heumann, M. Epple, "Transfection of cells with

custom-made calcium phosphate nanoparticles coated with DNA", Journal of Materials Chemistry (in press).

2. T. Welzel, W. Meyer-Zaika, M. Epple, "Continuous preparation of monodisperse functio-nalised calcium phosphate nanoparticles with adjustable crystallinity", Chemical Communications (2004) 1204-1205.


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MULTIFUNCTIONAL POLYMER-MINERAL COMPOSITE MATERIALS AS BIOACTIVE SCAFFOLDS FOR BONE TISSUE ENGINEERING

Kirstin Suck1, Evgenia. G. Vlakh, Evgeniy2. F. Panarin2, Tatiana B. Tennikova2 and

Cornelia Kasper1*

1Institute for Technical Chemistry, University of Hannover, Callinstr. 3, 30167 Hannover, Germany

2Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg, Russia

The main goal of this work is the development of an “intelligent” approach to construct multifunctional composite scaffolds for bone cell growth and differentiation. For this purpose, different kinds of mineral macroporous supports, water soluble aldehyde-containing copolymers of N-vinylpyrrolidone as well as different non-specific and biospecific ligands governing cell adhesion and growth have been used. The composite materials were tested initially in cell culture experiments.


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BIOMATERIALS AND OPHTALMOLOGICAL IMPLANTS BASED ON NANOCRYSTALLINE HYDROXYAPATITE

V.K. Tsuber, A.I. Kulak, L.A. Lesnikovich, I.V. Trofimova Institute of General and Inorganic Chemistry, National Academy of Sciences of Belarus,

Surganova st. 9, Minsk 220072 Belarus, E-mail: [email protected]

Hydroxyapatite (HA) Ca10(PO4)6(OH)2 is characterized by a unique biocompatibility and at present it is considered as a very prominent material for the medical application in maxilliofacial surgery, dentistry, orthopedy and ophthalmology. We have developed the method for the synthesis of nanocrystalline HA in aqueous solutions based on the chemical interaction of soluble calcium salts with (NH4)2HPO4 in alkaline medium [1], as a result HA gel may be obtained. Using different drying procedures, HA gel can be hardened with obtaining different solid products, e.g. loose powder with the particle size ranged from 63 to 500 µm or dense blocks. When the gel is dried at 40-60oC, the formation of xerogel in a form of solid conglomerates is observed. After dispersing this substance to a powder consisted of the species with a size ≤60 µm, its surface area comprises 150-200 m2/g. HA gel can be injected into the damaged sites of bone to repair bone tissues. It was also used for the impregnation of biomaterials made of the alumina ceramic foam with a pour size of about 250-450 µm with porosity 90-95% and pressing strength 2-5 MPa. As a result, light porous ophthalmologic implants imaged in figure have been obtained. Clinical tests of ophthalmologic implants were conducted with 26 patients and gave positive results.

Figure. The image of ophthalmologic implants.

We also used the HA powder annealed at 800-1100oC for plasma sputtering onto the metal (titanium, stainless steel) implants used in dentistry. Thin HA films deposited onto the polymeric matrix composed of polyvinyl alcohol and collodion (4% solution of colloxolinum in the mixture of ethanol and diethyl ether) can be used in dentistry and maxilliofacial surgery. As a compact material, HA was assayed in traumatology and orthopedy for the implantation into bone in order to substitute bone defects. Experimental investigations of the model break of cortical bone exhibited high biological activity of HA gel in the stimulation of osteogenesis and reparation of damaged bone tissue [3]. It is established that HA gel possesses enhanced biological activity in comparison with HA powder. The clinical trial of the HA-based medication “Osteogel” (I phase) confirmed the results of experimental studies of its specific activity and toxicity. This work was supported by the Belarusian Republican Foundation for Fundamental Researches. 1. Lesnikovich L.A., Kulak A.I., Trofimova I.V. et al. Method of hydroxyapatite powder preparation.

Belarusian Patent No 960244, published 30.09.98. 2. Lesnikovich L.A., Tsuber V.K., Kulak A.I., Trofimova I.V. et al. Method of bioactive

hydroxyapatite coatings coatication. Belarusian Application No 20040206, published 15.03.2004. 3. Lesnikovich L.A., Tsuber V.K., Trofimova I.V., Kulak A.I. et al. Hydroxyapatite: biomaterials and

drugs on the based // Science and innovations NAS of Belarus.-2003.-No5-6.-P.75-82


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A SINGLE DOSE OF ZOLEDRONIC ACID MARKEDLY DECREASES PARTICLE-INDUCED BONE RESORPTION

+*Wedemeyer C; *von Knoch M, **von Knoch F; *Pingsmann A; ***Hilken G; ****Sprecher C; *****Henschke F; *Saxler G; *Löer F;

+*Department of Orthopaedic Surgery, University of Duisburg-Essen, Essen, Germany. +49-201-4089-2161, [email protected]

Introduction

- The most common cause of implant failure in joint replacement is aseptic loosening due to particle-induced osteolysis. (1). The bisphosphonate alendronate has already been shown to prevent particle induced osteolysis and implant loosening in a canine model (2 ), in a rat model (3), and to prevent implant induced periimplant bone loss in total hip arthroplasty in man (4). The same drug has been used by Schwarz et al (5) in the particle-related murine calvarial osteolysis model. However, bisphosphonate had to be applied daily in both experiments. After the recent development of more potent bisphosphonates which allow a single dose application, we performed an investigationusing the more potent zoledronic acid administered as a single dose on particle-induced osteolysis. Zoledronic acid is a new generation, imidazole-containig, bisphosphonate that inhibits osteoclastic bone resorption. It is much more potent than other bisphosphonates at inhibiting 1, 25-dihydroxyvitamin D3-induced hypercalcemia in a rat model and calcium release in a vitro calvarial bone resorption assay (6). Materials and Methods A recently established murine calvarial model of UHMWPE particle-induced osteolysis (7) was applied in twenty-eight C57BL/J6 mice in accordance with the official guidelines and following approval by the University and the local government. Animals were equally randomized to four groups. In group I animals underwent sham surgery only, in group II animals were treated with ultra-high molecular weight polyethylene (UHMWPE) particles (about 6 x 106 particles), in group III animals were treated with particles and received additional Zoledronic acid (1-hydroxy-2- (1H-imidazol-1-yl) ethylidene]bisphosphonic acid, Novartis Pharma AG, Basle, Switzerland) at a dose of ∼ 120mg/kg of body weight directly after surgery, seven animals received Zoledronic acid 4 days after surgery (group IV) . This dose regime was in the range of the dosage recommended for application in humans (15-60 µg /kg of body weight). The group size of seven was determined in a power analysis indicating that for a two-sided test at

alpha=0.05 there was 95% power to detect a significant difference in the two groups. Fourteen days postoperatively, the animals were sacrificed in a CO2 chamber. Particles Commercially pure UHMWPE polyethylene particles were obtained from the manufacturer (Ceridust VP 3610, Clariant, Gersthofen, Germany). The particle size was described by the manufacturer as 50 % of the particles being smaller than 5 µm and 90 % smaller than 9 µm. Specimen retrieval and histotechnical processing Retrieved calvaria were processed utilizing undecalcified hard tissue techniques. Four µm sections were taken in the frontal plane centered over the area of particle deposition. Subsequently, these sections were stained with Giemsa and immunohistochemically for tartrate-specific acid phosphatase-positive (TRAP) osteoclasts. Histomorphological analysis The midline bone resorption area was determined in Giemsa stained sections by tracing the area of soft tissue including any bone resorption pits in the midline suture (5). In detail, using one microscopic field at a magnification of 10x, the regions of interest, i.e. non-osseous tissue area adjacent and in continuity with the midline suture was encircled by the operator; the area was recorded and calculated automatically by the software. The number of osteoclasts was determined by counting the number of TRAP - positive cells within this field. Of each available section the values were averaged per animal, and the average bone resorption area as well as the number of osteoclasts for each group were determined.

Results Bone resorption was 0.26 mm2 ± 0.09 mm2 in animals with particle implantation, 0.14 mm2 ± 0.05 mm2 in animals with particle implantation and Zoledronic acid treatment directly after surgery (p= 0.0047), and 0.15 mm2 ± 0.05 mm2 in animals with particle


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implantation and Zoledronic acid treatment on the fourth postoperative day (p= 0.006). The osteoclast number was 8.7 ± 2.8 in animals without particle implantation and 20 ± 4 in animals with particle implantation, compared to 9.2 ± 2.5 in animals with particle implantation and Zoledronic acid treatment directly after surgery (p= 0.0001), and 10.3 ± 1.4 in animals with particle implantation and Zoledronic acid treatment on the fourth postoperative day (p= 0.0004). Figs. 1-4: Typical calvarial histology group 1-4 (Giemsa staining)

Fig. 5: Number of Osteoclasts

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Conclusions In this experiment a single s.c. dose was given and was shown to be effective in preventing particle-related osteolysis. Single-dose i.v. treatment has been shown to be effective against other pathologic conditions in humans (8). The animal model used here to reflect particle-induced osteolysis around hip replacing implants takes place within the relatively short period of 2 weeks, whereas osteolysis in the clinical setting is a long-term problem occurring after 1 year or more. Despite this experimental shortcoming the murine calvarial osteolysis model remains an effective and established tool to screen for potential drugs to treat particle induced osteolysis. A loose implant has to be removed and replaced surgically. But the prevention of this course of the disease might be accomplished by the infrequent administration of a potent bisphosphonate such as ZA. According to these results ZA is effective both before particle-induced osteolysis starts to occur and even after it has occurred. Taken together, these findings indicate that a single dose of ZA might have a favorable effect on particle-induced osteolysis and might prevent its progression to implant loosening.

References 1. Willert HG et al. Clin Orthop 258:95-107, 1990. 2. Shanbag PJ et al. Clin Ortop 344:33-43, 1997. 3. Milett PJ et al. JBJS 84 – A(2):236-249, 2002 4. Vanesmaa PK et al. J Bone Miner Res 16:2126-2130, 2001. 5. Schwarz EM et al. J Orthop Res 18(3):472-480, 2000. 6. Green JR et al. J Bone Min Res 9:745-751, 2000. 7. von Knoch M et al. J Orthop Res Mar;22(2):237-43, 2004. 8. Reid et al. N Engl J Med 346(9):653-661,2002.

Acknowledgement: Supported by the German Science Foundation (grant KN 553/1) and Novartis Pharma, Germany. ** Department of Surgery, Kantonsspital Chur, Switzerland *** Central Animal Laboratory, University of Duisburg- Essen, Essen, Germany **** AO Research Institute, Clavadelerstrasse, Davos Platz, Switzerland ***** Pathologisches Institut am Johannisstift, Paderborn, Germany


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EXTENSIVE REGIONAL BONE FORMATION IN A MODEL OF PARTICLE

INDUCED OSTEOLYSIS AFTER TREATMENT WITH ZOLEDRONIC ACID

+*Wedemeyer C; **von Knoch F; *Pingsmann A; ***Hilken G;

****Sprecher C; *****Henschke F; *Saxler G; *Löer F; *von Knoch M

+*Department of Orthopaedic Surgery, University of Duisburg-Essen, Essen,

Germany. +49-201-4089-2161, [email protected]

Introduction Particle-induced osteolysis is the primary cause of aseptic loosening of total joint replacements. A consensus has emerged that the predominant process is one of cytokine production in response to phagocytosis of implant wear particles resulting in increased proliferation and differentiation of osteoclast precursors into mature osteoclasts. The increased bone mineral density in patients receiving bisphosphonates has been attributed to inhibition of osteoclasts and induction of their apoptosis (1). Previous studies have shown that bone resorption is modulated by osteoblasts interfering with bisphosphonates. In addition, bisphosphonates may have direct effects on osteoblast function. Bisphosphonates have been shown to enhance the recruitment of osteoblasts from among human bone marrow stromal cells (2), stimulate osteoblastic proliferation and maturation (3,4) and inhibit apoptosis of the osteoblast in vitro (5). Further, in vivo studies showed enhanced net bone growth into implant porosities (6) and during distraction osteogenesis (7) as well as a pronounced thickening of periprosthetic cortical bone after bisphosphonate treatment (8). In a particle-induced murine calvarial osteolysis model we previously showed that bone resorption can be markedly decreased by a single s.c. dose of zoledronic acid (9). Using the same model, we aimed to investigate the in vivo effects of zoledronic acid on osteoblastic bone formation under conditions of polyethylene particle-induced osteolysis. Materials and Methods A recently established murine calvarial model of ultra-high molecular weight polyethylene (UHMWPE) particle-induced osteolysis (10) was applied in twenty-eight C57BL/J6 mice in accordance with the official guidelines and following approval by the University and the local government. Animals were equally randomized to four groups. In group 1 animals underwent sham surgery only, in group 2 animals were treated with UHMWPE particles (about 6 x 106 particles), in group 3 animals were treated with particles and received

additional Zoledronic acid (Zometa, Novartis Pharma AG, Basle, Switzerland) at a dose of 25 mg/kg of body weight directly after surgery, seven animals received zoledronic acid 4 days after surgery (group 4) . This dose regime was in the range of the dosage recommended for application in humans. The group size of seven was determined in a power analysis. Statistical analysis was performed using a one-way ANOVA and a post-hoc Student’s two tailed t-test. All p-values were compared to an α-value of 0.05 to determine significance Particles Commercially pure UHMWPE polyethylene particles were obtained from the manufacturer (Ceridust VP 3610, Clariant, Gersthofen, Germany). The particle size was described by the manufacturer as 50 % of the particles being smaller than 5 µm and 90 % smaller than 9 µm. Specimen retrieval and histotechnical processing 14 days postoperatively, the animals were sacrificed in a CO2 chamber. Retrieved calvaria were processed utilizing undecalcified hard tissue techniques. Four µm sections were taken in the frontal plane centered over the area of particle deposition. These sections were stained with Giemsa. Histomorphometric analysis Bone formation was assessed by quantifying osteoid formation in Giemsa stained sections. Osteoid formation was determined by tracing the area of osteoid. In detail, using one microscopic field at a magnification of x10, the osteoid tissue area was encircled and the area was calculated automatically. Bone thickness was measured as an indicator of net bone growth. Then specimens were divided in four 0.5 mm measured steps to the left side and four measured steps to the right side of the midline suture. Bone thickness was measured successively at these measured steps.. Results UHMWPE particle burden without any further intervention (group 2) induced bone resorption to sham control levels (Figs. 1, 2). Additional treatment with Zoledronic acid significantly increased osteoblast`s activity and the osteoid


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formation (Figs. 3, 4) as quantitatively assessed by histomorphometry. Osteoid formation was 0.00 ± 0.00 mm² in sham group (group 1), 0.02 ± 0.03 mm² in animals with particle implantation (group 2), 0.25 ± 0.08 mm² in animals with particle implantation and Zoledronic acid treatment directly after surgery (group 3) (p= 0.0018), and 0.21 ± 0.11 mm² in animals with particle implantation and Zoledronic acid treatment on the fourth postoperative day (group 4) (p= 0.0042) (Fig. 5). The mean bone thickness was 0.2 ± 0.04 mm (range 0.17 to 0.31) in group 1 and 0.16 ± 0.02 mm (range 0.14 to 0.19) in group 2, 0.31 mm ± 0.04 mm (range 0.28 to 0.39) in group 3, and 0.29 mm ± 0.02 mm (range 0.28 to 0.34) in group 4. Student’s t-test revealed a statistical significant difference in mean calvarial bone thickness between group 2 and 3 (p= 0.00042), and group 2 and 4 (p= 0.0019). There was no statistical significant difference in bone thickness between group 3 and 4 (p= 0.07). Figs. 1-4: Typical calvarial histology group 1-4 (Giemsa staining)

Fig. 5: New bone formation in mm² (group 1-4)

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Conclusions In our previous experiment a single s.c. dose Zoledronic acid was given and shown to be effective in preventing particle-related osteolysis (9). Here we report that zoledronic acid further enhanced osteoblastic bone formation under conditions of particle-induced osteolysis. Consistent with previous studies (6-8), our results suggest that osteoblasts exposed to a single dose of zoledronic acid s.c. are able to increase their bone forming potential. The mechanisms by which they mediate their actions remain as yet unclear. The bisphosphonate-mediated effects of locally increased bone formation in a situation where bone may be excessively resorbed, e.g. due to wear debris, could be of significant benefit in order to ultimately improve the durability of total joint replacements.

References 9. Reid IR et al. N Engl J Med 346(9):653-61, 2002. 10. von Knoch F et al. Trans ORS 873, 2003. 11. Im G et al. Biomaterials 25:4105-15, 2004. 12. Reinholz GG et al. Cancer Res 60:6001-07, 2000. 13. Plotkin LI et al. J Clin Invest 104:1363-1374, 1999. 14. Shanbhag et al.Trans ORS: 93, 1999. 15. Little DG et al. J Bone Miner Res 18; 1300-1307, 2003. 16. von Knoch F et al. Trans ORS 1375, 2003. 17. von Knoch M et al. Biomaterials 2004 in press. 18. von Knoch M et al. J Orthop Res Mar;22(2):237-43, Acknowledgement: Supported by the German Science Foundation (grant KN 553/1) and Novartis Pharma, Germany.

** Department of Surgery, Kantonsspital Chur, Switzerland

*** Central Animal Laboratory, University of Duisburg- Essen, Essen, Germany

**** AO Research Institute, Clavadelerstrasse, Davos Platz, Switzerland

***** Pathologisches Institut am Johannisstift, Paderborn, Germany


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INTELLIGENT BIOREACTOR FOR TISSUE CULTIVATION

K.Ch.Wierzcholski,K.Kosowski

Technical University Gdańsk, Faculty of Ocean Engineering& Ship Technology,Narutowicza 11 PL-80952 Gdańsk

ABSTRACT

The WHO has declared the present decade the bone and joint decade. In 1988 in USA the estimated total cost of osteoarthrosis was $54,6 billion. Annually in Germany more than 30 million medical treatments and more than 50 million days of temporary or permanent disablement, are due the diseases of cartilage and bone. It is not surprising that the number of human joint diseases shows an exponential increase over the last years in Europe. In last ten years in European Union the number of bone fractures caused by the osteoarthritis increased twofold. More than 100000 total hip or knee joints are implanted during one year in Germany. Within ten years 5% of them fail by aseptic loosening.It can be estimated that yearly 2.5 million joint fractures take place worldwide. Only in 2000 in European Union 414 000 joint fractures was recorded. The increase in number of joint fractures in European Union, anticipated on the ground of demographic development in the next 50 years, would reach about one million.

The tissue cultivation and engineering of cartilage implants in bioreactor may open a new path for the surgical treatment of joint surface defect. Autologous chondrocyte transplantation has been gaining in clinical significance over the last several years. The tissue engineering of cartilage implants of human joints is of a great importance for treatments - by transplantation - of various cartilage diseases. The proposed paper is also addressed to such social objectives as quality of life, human health and professional safety. Intrinsic mechanism of cartilage regeneration and cartilage repair will be also investigated in the proposed paper. The articular cartilage is considered a tissue without the capacity to repair, thus mechanical wear of tissue joint is often designated as a normal tribute to increasing age. Therefore cultivation of natural human tissues in bioreactor is really very important problem.

Intelligent bioreactor is designed as an interdisciplinary, multi-stage, analytical, numerical and experimental measure whose ultimate goal is to develop a prototype of intelligent bioreactor for tissue cultivation and joint cartilage repair and remodeling. The proposal will deliver two main outputs:

1. New generation of nutrient fluid by applying the ferrofluid additions and a new method of perfusion and simultaneously control of the tissue growth using the viscosity control method,

2. Intelligent bioreactor which can “remember” the process of cartilage (tissue) cultivation for “learning” and implementation of optimal control system based on the created knowledge base by applying the neuro-fuzzy methodology. The idea of “intelligent bioreactor” should be treated not only as a mechanical-biological process, but also as a process which improves the quality of cultivating tissue leading to development of characteristics of extracted biomaterial.

The objective of this paper is to modeling, simulation, control and optimization of the tissue behaviour during cultivating process in a bioreactor, leading to elaboration of the prototype of an intelligent bioreactor by using advanced optimization algorithms such as neuro-fuzzy methods.

Intelligent bioreactor for cartilage cultivation enables utilization of radically innovative technologies and can lead to enormous benefits caused by the multifunctional cultivation. The paper should focus on technological novelties and methods applied during the cultivation with relation to chemical and physical processes.

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