Roman Major

Self-assembling surfaces of blood-contacting materials

The optimal scaffold should have the self-organising property of activating the appropriate tissues surrounding the re-population. The anti-bacterial property of the coating was obtained through surface pre-treatment with coatings a few nanometres in thickness deposited using vapour-based methods. The coating’s anti-thrombogenic properties were obtained by the selective mobilisation of cellular functions, which was controlled by the structure of porous coatings deposited on bulk substrates and by the small biological agent-l-arginyl-glycyl-l-aspartic acid (tripeptide Arg-Gly-Asp-RGD) protein domains. Two tests simulating arterial flow conditions were performed: Impact-R, for examining platelet function under near physiological conditions, and radial flow chamber, a cell detachment test that gives an overview of cell behaviour and shear stresses that could appear between the cell and the biomaterial. Cell structures were analysed using laser scanning confocal microscopy and flow cytometry. The performed in vitro dynamic test for the haemo-compatibility revealed the most promising surface functionalization was based on porous extracellular-like structure covered with endothelium cells simultaneously. The antibacterial function was achieved by the appropriate phase composition of the coating used for the pre-treatment stage. The coating for the pre-treatment was selected on the basis of the blood-material and bacteria-material interaction.

Thrombogenicity and biocompatibility studies of reduced graphene oxide modified acellular pulmonary valve tissue

Current strategies in tissue engineering seek to obtain a functional tissue analogue by either seeding acellular scaffolds with cells ex vivo or repopulating them with cells in vivo, after implantation in patients. To function properly, the scaffold should be non-thrombogenic and biocompatible. Especially for the case of in vivo cell repopulation, the scaffold should be prepared in a manner that protects the tissue against platelet activation and adhesion. Anti-thrombogenicity can be achieved by chemical or physical surface modification. The aim of our study was to evaluate the platelet activation and thrombogenic properties of an acellular tissue scaffold that was surface modified with reduced graphene oxide (rGO). Graphene oxide was prepared by a modified Hummers method. For the study, an acellular pulmonary valve conduit modified with rGO was used. The rGO modified tissue samples were subjected to in vitro testing through interaction with whole blood under simulated laminar flow conditions. The following cellular receptors were then analysed: CD42a, CD42b, CD41a, CD40, CD65P and PAC-1. In parallel, the adhesion of platelets (CD62P positive), leukocytes (CD45 positive) and platelet-leukocyte aggregates (CD62P/CD45 positive) on the modified surface was evaluated. As a reference, non-coated acellular tissue, Poly-l lysine and fibronectin coated tissue were also tested. The rGO surface was also analysed for biocompatibility by performing a cytotoxicity test, TUNEL assay and Cell Cycle analysis. There was no significant difference in platelet activation and adhesion between the study groups. The only significant difference was observed for the PAC-1 receptor between Poly-l lysine group and rGO and the percentage of PAC-1 positive cells was 6% and 18% respectively. The average number of activated platelets (CD62P) in the field of view was 1, while the average number of leukocytes in the field of view was 3. No adherent platelet-leukocyte aggregates were observed. There were no significant differences in the DNA fragmentation. No significant effect of rGO on the amount of cells in different phases of the cell cycle was observed. Cytotoxicity indicates that the rGO can damage cells in direct contact but have no effect on the viability of fibroblasts in indirect contact.

Hemocompatible, pulsed laser deposited coatings on polymers / Anwendung der Pulslaserbeschichtung zur Abscheidung von hämokompatiblen Beschichtungen auf Polymeroberflächen

Abstract State-of-the-art non-thrombogenic blood contacting surfaces are based on heparin and struggle with the problem of bleeding. However, appropriate blood flow characteristics are essential for clinical application. Thus, there is increasing demand to develop new coating materials for improved human body acceptance. Materials deposited by vacuum coating techniques would be an excellent alternative if the coating temperatures can be kept low because of the applied substrate materials of low temperature resistance (polymers). Most of the recently used plasma-based deposition techniques cannot fulfill this demand. However, adequate film structure and high adhesion can be reached by the pulsed laser deposition at room temperature, which was developed to an industrial-scaled process at Laser Center Leoben. Here, this process is described in detail and the resulting structural film properties are shown for titanium, titanium nitride, titanium carbonitride, and diamond-like carbon on polyurethane, titanium and silicon substrates. Additionally, we present the biological response of blood cells and the kinetic mechanism of eukaryote cell attachment. In conclusion, high biological acceptance and distinct differences for the critical delamination shear stress were found for the coatings, indicating higher adhesion at higher carbon contents. Zusammenfassung Derzeit verwendete Oberflächen für den Blutkontakt basieren im Allgemeinen auf Heparin, wobei es in der klinischen Verwendung leicht zum Problem von Blutungen kommen kann. Nichtsdestotrotz ist aber ein optimaler Blutfluss entscheidend, wodurch sich zunehmend die Forderung nach der Entwicklung von neuen Materialien mit verbesserter Körperakzeptanz stellt. Mittels Plasmaverfahren auf Oberflächen abgeschiedene Werkstoffe könnten zukünftig eine Alternative zu Heparin bieten, wenn die Temperaturen während des Herstellprozesses auf nahezu Raumtemperatur abgesenkt werden könnten, um vor allem die vielfach verwendeten Kunststoffe mit geringer Temperaturbeständigkeit beschichten zu können. Als eine der wenigen Techniken bietet sich dafür die Pulslaserabscheidung (PLD) an, welche zu einem industriell einsetzbaren Prozess am Laserzentrum Leoben entwickelt wurde. Diese Arbeit beschreibt die Hauptmerkmale dieses Prozesses und die sich ergebenden strukturellen Filmeigenschaften von Ti, TiN, Ti(C,N) und diamantähnlichem Kohlenstoff (DLC) auf Polyurethan-, Titan- und Siliziumoberflächen. Zudem werden die biologische Antwort von Blutzellen und die kinetischen Haftungsmechanismen von Eukaryotzellen dargestellt. Zusammengefasst zeigen diese Schichtwerkstoffe hohe biologische Akzeptanz und deutliche Unterschiede in den kritischen Scherspannungen zur Delamination, wobei eine höhere Zelladhäsion bei höheren Kohlenstoffgehalten erreicht wird.

Hemocompatibility of Inorganic Physical Vapor Deposition (PVD) Coatings on Thermoplastic Polyurethane Polymers

Biocompatibility improvements for blood contacting materials are of increasing interest for implanted devices and interventional tools. The current study focuses on inorganic (titanium, titanium nitride, titanium oxide) as well as diamond-like carbon (DLC) coating materials on polymer surfaces (thermoplastic polyurethane), deposited by magnetron sputtering und pulsed laser deposition at room temperature. DLC was used pure (a-C:H) as well as doped with silicon, titanium, and nitrogen + titanium (a-C:H:Si, a-C:H:Ti, a-C:H:N:Ti). In-vitro testing of the hemocompatibility requires mandatory dynamic test conditions to simulate in-vivo conditions, e.g., realized by a cone-and-plate analyzer. In such tests, titanium- and nitrogen-doped DLC and titanium nitride were found to be optimally anti-thrombotic and better than state-of-the-art polyurethane polymers. This is mainly due to the low tendency to platelet microparticle formation, a high content of remaining platelets in the whole blood after testing and low concentration of platelet activation and aggregation markers. Comparing this result to shear-flow induced cell motility tests with e.g., Dictostelium discoideum cell model organism reveals similar tendencies for the investigated materials.

Surface treatment of thin-film materials to allow dialogue between endothelial and smooth muscle cells and the effective inhibition of platelet activation

This work aims to reproduce the structure of a blood vessel. The surface modifications that were carried out concerned the creation of an appropriate environment for communication between tissues. Cell–material interactions were studied in this work. Samples for examination were prepared on polished silicon substrates and the sample surface was covered with different biocompatible coatings such as: diamond like carbon (a-C:H), titanium (Ti), titanium nitride (Ti:N), titanium carbonitride (a-C:H:Ti:N), titanium oxide (Ti:O), silicon doped diamond like carbon (a-C:H:Si) and titanium doped diamond like carbon (a-C:H:Ti). Physical vapour deposition (hybrid pulsed laser deposition) was chosen for coating fabrication and the coating thickness ranged from 200 nm to 1 μm. Based on a dynamic analysis using blood, which considered aggregate formation, platelet consumption and blood platelet activation, the most haemocompatible coating was chosen. The study also considered channels for the migration of cells. Using the laser ablation method, migration channels were fabricated. The width of each individual channel depended on the cell size, which was close to 10–30 μm. Cells were deposited directly on the migration channels. An influence of the heat affected zone on cell proliferation was observed. Porous coatings were deposited as the next step after channel formation. The coatings were stabilized by a cross linking chemical reaction using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. Smooth muscle cells were deposited on the surface of samples without the porous coating, directly onto the migration channels. Human umbilical endothelial cells were deposited on the top of the synthetic porous coating. A de novo surface design for cardiovascular purposes with the function of providing oriented cell growth as well as allowing a dynamic dialogue between tissues was proposed.

Inner surface modification of the tube-like elements for medical applications

The objective of this work was to modify the inner surfaces of polymer tubes to improve their biocompatibility with blood cells. New materials that were designed to be placed in contact with blood during forced blood circulation were studied. The inner surfaces of these tubes were covered with anti-thrombogenic coatings. Materials based on silicon carbide, silicon oxide, and silicon nitride were deposited. Depending on the topography and the chemical nature of the inner vessel surface, a non-thrombogenic bio-surface, also called a neointima, was formed. Several expected applications of this work are discussed, including archetypal human blood vessels, the design of a nanostructural artificial substitute, optimising the surface using vacuum-coating techniques, characterising materials on multiple scales, and studying the blood-material interaction under dynamic conditions in an arterial flow environment. Several promising solutions for inhibiting the activation of the blood-clotting cascade via the use of appropriate surface architectures have been obtained. These solutions may be applied in advanced biocompatible cardiovascular implants.

In vitro hemocompatibility on thin ceramic and hydrogel films deposited on polymer substrate performed in arterial flow conditions.

Hydrogel coatings were stabilized by titanium carbonitride a-C:H:Ti:N buffer layers deposited directly onto the polyurethane (PU) substrate beneath a final hydrogel coating. Coatings of a-C:H:Ti:N were deposited using a hybrid method of pulsed laser deposition (PLD) and magnetron sputtering (MS) under high vacuum conditions. The influence of the buffer a-C:H:Ti:N layer on the hydrogel coating was analysed by means of a multi-scale microstructure study. Mechanical tests were performed at an indentation load of 5 mN using Berkovich indenter geometry. Haemocompatible analyses were performed in vitro using a blood flow simulator. The blood-material interaction was analysed under dynamic conditions. The coating fabrication procedure improved the coating stability due to the deposition of the amorphous titanium carbonitride buffer layer.

Effects of the surface modification of polyurethane substrates on genotoxicity and blood activation processes

The aim of this study was to determine the mutagenic and thrombogenic potential of a material composed of a thin coating deposited on a polymeric substrate. In this work, a surface was modified in a manner that would mimic the function of cellular niches. Finally, the surfaces should actively capture and differentiate progenitor cells from the blood stream. Thin films with 10 to 500nm thicknesses were deposited by unbalanced, pulsed DC magnetron sputtering on smooth polyurethane. Such high energy conditions led to a stiffening of the polymer surface layers by pseudodiffusion during the initial stages of film growth. Both the high intrinsic film stress due to high energy film growth and the huge difference in the elastic properties of the films and polymer substrates resulted in hierarchical and self-adapting nanowrinkling. Surface modifications of synthetic materials for future use in regeneration of the circulatory system must be tested in terms of their thrombogenicity and mutagenicity. Point mutations in many cases can lead to many serious haematologic complications. Genotoxicity was determined by testing for reverse histidine mutations in selected strains of Salmonella typhimurium. The analysis was performed in the presence and absence of metabolic activation system S9 containing liver microsomal fraction of rats. Based on these results, no mutagenicity of the tested material was observed. The interaction of blood and the material under dynamic conditions was described. Blood from above the analysed surface was collected after the test, and the quality of the blood was assessed along with the type of cellular response to the surface. In the obtained results of the coagulation processes, it was found that the tested material reduced the process of platelet activation under hydrodynamic conditions in comparison to the control material, polyurethane.

Effect of the silicon carbide nanoparticles introduction on biological properties of porous polymer coatings

The multilayer polyelectrolyte films (PEMs) seem to be promising coatings to simulate the structure and behavior of the extracellular matrix. PEMs constructed through Layer by Layer deposition of oppositely charged polymers have become a powerful tool for tailoring biointerfaces. Films consisting of chitosan/chondroitin sulfate polymers exhibit a fast biodegradability in the environment of human tissues. Lifetime extension of this material type could be implemented by its structure stabilization through cross-linking or introduction of nanoparticles. Transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM) methods were used to determine the microstructure and localization of the silicon carbide nanoparticles introduced to the extracellular like structure of the polymer coatings. The numerical analysis of nanoparticles dispersion and mechanical properties was verified by indentation measurements. Modified coatings biocompatibility was analyzed by cytotoxicity assays and microscopic observations of the growth of endothelial cells on the material surface. Comparison of stabilization methods including chemical cross-linking and SiC nanoparticles introduction into multilayer polyelectrolyte films has shown that both stabilizers could be useful for biomedical applications. However, SiC nanoparticles application could be limited by slightly lower endothelialization efficiency and risk of cytotoxicity due to their release from coatings.

Industrially-scaled pulsed laser deposition based coating techniques for the realization of hemocompatible surfaces for blood contact applications

Non-thrombogenic blood contacting surfaces and appropriate blood flow characteristics are essential for clinical application. State-of-the-art coatings are based on heparin and struggle with the problem of bleeding. Thus, there is increasing demand for developing new coating materials for improved human body acceptance. Materials deposited by vacuum coating techniques would be an excellent alternative if the coating temperatures can be kept low due to the applied substrate materials of low temperature resistance (mostly polymers). Under these circumstances, adequate film structure and high adhesion can be reached by the Pulsed Laser Deposition at room temperature (RT-PLD), which was developed to an industrial-scaled process at Laser Center Leoben. This process was applied to deposit Ti, TiN, TiCN and diamond-like carbon (DLC) on polyurethane, titanium and silicon substrates to study the biological interactions to blood cells and the kinetic mechanism of eukaryote cell attachment. Besides high biological acceptance, distinct differences for the critical delamination shear stress were found for the coatings, indicating higher adhesion at higher carbon contents.