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1

Biomechanical properties of the thin PVD coatings defined by red blood cells

Trembecka-Wojciga K., Major R., Lackner J. M., Bruckert F., Jasek E., Major B.

Bulletin of the Polish Academy of Sciences. Technical Sciences

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2015

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Vol. 63, nr 3

697--705

EN

The measurement of the strength of bonds between biomaterials and cells is a major challenge in biotribology since it allows for the identification of different species in adhesion phenomena. Biomaterials, such as diamond-like carbon (DLC), titanium, and titanium nitride, seem to be good candidates for future blood-contact applications. These materials were deposited as thin films by the hybrid pulsed laser deposition (PLD) technique to examine the influence of such surfaces on cell behavior. The biomaterial examinations were performed in static conditions with red blood cells and then subjected to a dynamical test to observe the cell detachment kinetics. The tests revealed differences in behavior with respect to the applied coating material. The strongest cell-biomaterial interaction was observed for the carbon-based materials compared to the titanium and titanium nitride. Among many tests, a radial flow interaction analysis gives the opportunity to analyze cell adhesion to the applied material with the high accuracy. Analysis of concentrates helped to select materials for further dynamic tests on blood using an aortic flow simulator. In this case, the platelet adhesion to the surface and their degree of activation was analyzed. The quality of the selected coating was tested using a scratch test. The analyses of the microstructure were done using high resolution transmission electron microscopy. The phase composition and the residual stress were analyzed using X-ray diffraction methods.

2

Computer engineering in designing and fabrication of tissue analogue-type coating dedicated for the cardiovascular regeneration

Trembecka-Wojciga K., Major R., Bruckert F., Lackner J. M., Lacki P., Sanak M., Major B.

Archives of Civil and Mechanical Engineering

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2015

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Vol. 15, no. 3

621--630

EN

The work was related to the development of novel methods in designing and fabrication of thin, porous, tissue-like coatings. The surface modification was designed to create an environment for the appropriate cell growth. The originally designed system was established to prepare porous, synthetic coatings. The dedicated software was elaborated to control the sequential coating deposition based on the electrostatic interaction. The finite elements method (FEM) was used to determine structural and mechanical properties of the coatings. The numerical model was verified experimentally. The performed simulation predicted the coating stabilization by the graphene nanoparticles. Graphene was introduced as a stabilizer of the polymer coating. The elaborated automatic system allowed preparation the porous coatings, repetitively. Coatings were stabilized by the cross-linking chemical reaction using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. Nanoparticles were introduced by means of the electrostatic interaction. Mechanical analysis revealed an influence of the porous structure modification on the coating stiffness. Dynamic tests on blood subjected to the aortic flow showed antithrombogenic properties of the elaborated coatings.

3

Graphene based porous coatings with antibacterial and antithrombogenous function - Materials and design

Major R., Sanak M., Mzyk A., Lipinska L., Kot M., Lacki P., Bruckert F., Major B.

Archives of Civil and Mechanical Engineering

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2014

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Vol. 14, no. 4

540--549

EN

The studies considered graphene-based biomaterials dedicated for cardiovascular therapy. Reduced graphene oxide flakes were introduced into the porous structure of the polyelectrolyte based coatings. TEM analysis showed the presence of graphene flakes arranged parallel to the substrate surface, firmly connected to the porous coating. Biomaterials were subjected to a comprehensive diagnosis of the biological and material properties. The material behavior was simulated using finite element method. The coatings were deposited using layer by layer method. Mechanical analysis was done using Berkovich indenter geometry. They confirmed theoretical FEA based calculations, it was observed the coating stiffness incensement under the influence of introduced particles of graphene. The endanger of the bacteriology film formation was verified based on the E-coli and Staphylo coccus bacteria. Blood–material interaction was examined in the dynamic flow conditions. Bacteriological analysis showed reduced presence of bacteria after contact with the surface with introduced graphene flakes. Dynamic analyzes on blood showed high activation of the coagulation, strong platelets consumption and a strong immune response. It is caused by sharp edge of the single plane of the graphene flake.

4

Protein conformational changes induced by adsorption onto material surfaces: an important issue for biomedical applications of material science

Ballet T., Boulange L., Brechet Y., Bruckert F., Weidenhaupt M.

Bulletin of the Polish Academy of Sciences. Technical Sciences

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2010

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Vol. 58, nr 2

303-315

EN

Protein adsorption on solid surfaces is a widespread phenomenon of large biological and biotechnological significance. Conformational changes are likely to accompany protein adsorption, but are difficult to evidence directly. Nevertheless they have important consequences, since the partial unfolding of protein domains can expose hitherto hidden amino acids. This remodeling of the protein surface can trigger the activation of molecular complexes such as the blood coagulation cascade or the innate immune complement system. In the case of extracellular matrix, it can also change the way cells interact with the material surfaces and result in modified cell behavior. In this review, we present direct and indirect evidences that support the view that some proteins change their conformation upon adsorption. We also show that both physical and chemical methods are needed to study the extent and kinetics of protein conformational changes. In particular, AFM techniques and cryo-electron microscopy provide useful and complementary information. We then review the chemical and topological features of both proteins and material surfaces in relation with protein adsorption. Mutating key amino acids in proteins changes their stability and this is related to material-induced conformational changes, as shown for instance with insulin. In addition, combinatorial methods should provide valuable information about peptide or antibody adsorption on well-defined material surfaces. These techniques could be combined with molecular modeling methods to decipher the rules governing conformational changes associated with protein adsorption.

5

Nanostrukturalne powłoki na bazie tytanu do kontaktu z krwią; diagnostyka strukturalna, adhezja komórek w warunkach hydrodynamicznych

Major R., Bruckert F., Lackner J. M., Kustosz R., Lacki P., Major B.

Inżynieria Materiałowa

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2009

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Vol. 30, nr 5

356-358

PL

Adhezja i aktywacja komórek krwi do powierzchni materiałów o przeznaczeniu na implanty naczyniowe jest bardzo istotnym zagadnieniem. Parametry strukturalne istotnie wpływają na interakcję biomateriału z tkanką łączną. Celem prowadzonych prac jest wytworzenie gradientowego materiału o odpowiednich właściwościach strukturalnych i dobrej biozgodności. Przedmiotem realizowanych badań są cienkie warstwy gradientowe na bazie tytanu metalicznego oraz stechiometrycznego TiN i Ti(C, N) nanoszone róż-nymi metodami, a mianowicie: ablacji laserowej (PLD), magnetronową oraz hybrydową łączącą PLD z magnetronową na podłoża tak metaliczne (tytanowe) oraz na klinicznie stosowany poliuretan. Proces osadzania realizowano w Centrum Laserowym w Austrii, kompleksową diagnostykę strukturalną na podstawie badań XRD, SEM, TEM, HRTEM w IMIM PAN w Krakowie. W ośrodku francuskim w Laboratorium Biofizycznym na Politechnice w Grenoble oraz Fundacji Rozwoju Kardiochirurgii w Zabrzu prowadzono testy wyznaczenia kinetyki przylegania modelowych komórek do materiałów o potencjalnym zastosowaniu biomedycznym. Skonstruowano unikatową aparaturę do badań adhezji komórek w warunkach przepływu medium. Analiza prowadzona jest na podstawie badań komórek eukariota, której prosty genom jest łatwo manipulowany przez techniki biologii molekularnej. Dane doświadczalne uzyskuje się na drodze analizy obrazów fluorescencyjnych po przeprowadzonym teście w warunkach hydrodynamicznych i służą one do wyznaczania krzywych kinetycznych. Kompleksowa diagnostyka strukturalna realizowana jest metodą rentgenografii strukturalnej oraz skaningowej i transmisyjnej mikroskopii elektronowej i ma na celu opis morfologii powierzchni, analizę właściwości fizycznych oraz mechanicznych. Uzyskane wyniki z przeprowadzonych testów modelowych stanowić będą podstawę do testów kinetyki z zastosowaniem komórek krwi i określenia ich interakcji ze sztuczną powierzchnią. Problem agregacji płytek krwi do sztucznych powierzchni wprowadzanych do organizmu pacjenta jest bardzo istotny w realizowanej we współpracy z Fundacją Rozwoju Kardiochirurgii w Zabrzu problematyce związanej z wieloletnim projektem strategicznym "Polskie Sztuczne Serce".

EN

The measurement of the strength of bonds between biomaterials and cells is a major challenge in cellular biology since it allows for the identification of different species in adhesion phenomena. Biomaterials, such as diamond-like carbon (DLC), titanium (Ti), and stoichiometric titanium nitride (TiN) as well as titanium carbo-nitrade (Ti(C, N)), seem to be good candidates for future blood-contact applications. These materials were deposited as thin films by the hybrid pulsed laser deposition (PLD) technique to examine the influence of such surfaces on cell behaviour. The kinetic energy of the evaporated particles was controlled by application of different reactive and non reactive atmospheres during deposition. High resolution transmission electron microscopy (HRTEM) was used to reveal structure dependence on specific atmosphere in the reactive chamber. New experimental technique to examine the crystallographic orientation based on X-ray texture tomography was applied to estimate contribution of the atmosphere to crystal orientation. The biomaterial examinations were performed in static conditions with Dictyostelium discoideum cells and then subjected to a dynamical test to ob-serve the cell detachment kinetics. For a given cell, detachment occurs for critical stress values caused by the applied hydrodynamic pressure above a threshold which depends on cell size and physicochemical properties of the substrate, but it is not affected by depolymerization of the actin and tubulin cytoskeleton. Tests revealed differences in behavior in respect to the applied coating material. The strongest cell-biomaterial interaction was observed for the carbon-based materials compared to the titanium and titanium nitride. Correlations between the cell adhesion and surface properties of materials were examined using the texture tomography and the surface topography techniques. A finite element modelling exhibited cell reactions to shear stress impact. A problem of blood plates adhesion to artificial surfaces implanted into a patient body is of a crucial type in realization of the multiyear strategic project "Polish Artificial Heart".

6

Kinetics of eukaryote cells adhesion under shear flow detachment on the PLD deposited surfaces

Major R., Bruckert F., Lackner J.M., Waldhauser W., Pietrzyk M.

Bulletin of the Polish Academy of Sciences. Technical Sciences

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2008

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Vol. 56, nr 3

223-228

EN

Hybryd PLD method was used for deposition high quality thin Ti, TiN, Ti(C,N) and DLC coatings. The kinetic energy of the evaporated particles was controlled by application of variation of different reactive and non reactive atmospheres during deposition. The purpose was to improve adhesion by building a bridge between the real ceramic coating and the substrate. A new layer composition layout was proposed by application of a buffer, starting layer. Advanced HRTEM investigation based on high resolution transmission electron microscopy was used to reveal structure dependence on specific atmosphere in the reactive chamber. New experimental technique to examine the crystallographic orientation based on X-ray texture tomography was applied to estimate contribution of the atmosphere to crystal orientation. Using Dictyostelium discoideum cells as a model organism for specific and nonspecific adhesion, kinetics of shear flow-induced cell detachment was studied. For a given cell, detachment occurs for critical stress values caused by the applied hydrodynamic pressure above a threshold. Cells are then removed from the substrate with an apparent first-order rate reaction that strongly depends on the stress. The threshold stress depends on cell size and physicochemical properties of the substrate, but it is not affected by depolymerization of the actin and tubulin cytoskeleton.

7

Coatings on TiN and Ti(C,N) basis for biomedical application to blood contact and TiN/CrN multilayered tribological systems produced by pulsed laser deposition

Major B., Bruckert F., Lackner J. M., Ebner R., Kustosz R., Lacki P.

Archives of Metallurgy and Materials

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2008

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Vol. 53, iss. 1

39-48

EN

Research activity on surface en g ineering performed at the IMIM PAS in last years has been presented. Experiments were focused on TiN and Ti(C,N) thin coatings produced on titanium and biologically applied polyurethane substrates by application of pulsed laser deposition (PLD), magnetron sputtering (MS), and hybrid PLD/MS, at room temperature. Bio-physical tests of the kinetics of shear flow-induced cell detachment have been carried out. Model eucariotic cells easy to manipulate using technics of molecular biology have been used in experiments. Fluorescence patterns obtained after the performed test at kinetics condition tests have been used to establish kinetics curves. A microstructure examination by application of XRD and transmission electron microscopy have been performed. On the basis of the real cell detachment experiment, a finite element simulation was done. The highest shear stress was estimated for the region of the radius of the whole pierced in the center of the upper disc. There are an increasing number of applications in tribology where the properties of a sin gle material are not sufficient. One way to surmount this problem is to use a multilayer coating. Application of metallic interlayers improves adhesion of nitride hard layer in multilayer systems. Tribological coatings consisted of 4, 8 and 32 layers of Cr/CrN and Ti/TiN types were fabricated with the PLD technique. It is found in transmission electron examinations on thin foils prepared from cross-section that both nitride-based multilayer structures studied are characterized by small columnar crystallite sizes and high defect density, what might raise their hardness but compromise coating adhesion. The intermediate metallic layers contained larger sized and less defective columnar structure compared to the nitride layers located at close to the substrate which should improve the coatings toughness. Switching from single layer to multi-layer metal/nitride composition improved resistance to delamination.

PL

Przedstawiono działalność IMIM PAN prowadzoną w ostatnich latach w zakresie inżynierii powierzchni. Badania koncentrowaly się na cienkich warstwach typu TiN i Ti(C,N) wytwarzanych z wykorzystaniem metody osadzania laserem impulsowym (PLD), magnetronowej (MS) oraz hybrydowej PLD/MS w temperaturze pokojowej na podłożu metalicznego tytanu oraz stosowanego biologicznie poliuretanu. Przeprowadzono testy biofizyczne kinetyki wymywania komórek w warunkach naprężenia ścinania generowanego przepływem. W badaniach zastosowano modelowe komórki eukariota, które są dogodne do wykorzystania w eksperymentalnych technikach biologii molekularnej. Obrazy fluorescencyjne uzyskane po przeprowadzonych testach w warunkach kinetycznych wykorzystane zostaly do wyznaczenia krzywych kinetycznych. Przeprowadzono badania mikrostruktury metodą dyfrakcji rentgenowskiej i transmisyjnej mikroskopii elektronowej. Wykorzystując wyniki rzeczywistego eksperymentu wymywania komórek, przeprowadzono modelowanie metodą elementów skończonych. Najwyższe naprężenia ścinające uzyskano dla obszaru leżącego na brzegu otworu centralnego górnego dysku. Wzrasta ilość zastosowań w tribologii gdy właściwości tworzywa jednowarstwowego są niewystarczające. Jednym z rozwiązań jest wytwarzanie powłok wielowarstwowych. Zastosowanie wielowarstwy metalicznej w układzie Ti/TiN, zawierającym twardą warstwę azotkową, poprawia adhezje. Wytworzone zostały metodą PLD układy tribologiczne składające się z 4, 8 oraz 32 warstw typu Cr/CrN i Ti/TiN. Stwierdzono w badaniach metodą transmisyjnej mikroskopii elektronowej na cienkich foliach uzyskanych z przekroju poprzecznego, że obydwa wielowarstwowe systemy charakteryzują się drobnokrystaliczną strukturą o małej wielkości ziaren kolumnowych z dużą gęstością defektów. Może to wpływać na wzrost twardości, obniżając jednak adhezję powłoki. Międzywarstwy metaliczne zawierają nieco większych rozmiarów i mniej zdefektowaną strukturę kolumnową niż warstwy azotkowe. Warstwa metaliczna jest pierwsząwarstwąprzy podłożu i podnosi ona plastyczność powłoki. Przechodząc od warstwy pojedynczej do wielowarstwy typu metal/azotek uzyskuje siępoprawę odporności na delaminację.

8

New gradient coatings on TiN and TiCN basis for biomedical application to blood contact

Major B., Major R., Bruckert F., Lackner J.M., Ebner R., Kustosz R., Lacki P.

Advances in Materials Science

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2007

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Vol. 7, nr 3(13)

63-70

EN

Adhesion of blood cells and its activation to material surface used as implants is a problem of crucial importance. Structure parameters influence essentially the interaction of biomedical materials with a human connective tissue. The work is focused on TiN and TiCN thin coatings produced on titanium substrate by application of different methods i.e. pulsed laser deposition (PLD), magnetron sputtering, and hybrid PLD, at room temperature. Biophysical tests of the kinetics of shear flow-induced cell detachment have been carried out. Model eucariotic cells easy to manipulate using technics of molecular biology have been used in experiments. Fluorescence patterns obtained after the performed test at kinetics condition tests have been used to establish kinetics curves. A microstructure examination by application of XRD and transmission electron microscopy have been performed. The work is realized in cooperation with the Foundation of Cardiac Surgery Development in the frame-work of partially implanted prosthesis of the artificial heart project.