Biomimmetic surfaces effectively inhibiting coagulation ... · journal eage: ww.necentralress ... The work focused on developing coatings with niche-like surfaces that resemble stem

REGENERATIVE NANOMEDICINE

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A B S T R AC T

Roman Majora,*, Klaudia Trembecka-Wojcigaa, Juergen Markus Lacknerb, Hanna Pluteckac, Boguslaw Majora, Stanislaw Miturad

Biomimmetic surfaces effectively inhibiting coagulation process dedicated for cardiovascular devices

The work focused on developing coatings with niche-like surfaces that resemble stem cells. Structures similar to stem cell niches were designed to inhibit the fast division process and subsequently to influence the efficient endothelialisation. Using a plasma method, the surface topography and stiffness, as well as the microstructure, were modified by controlled residual stresses. The cells exhibited a potential for formation of the appropriate endothelial layer. Residual stresses affected the formation of the surface topography in a form of nano-wrinkles similar to niches in the tissue environment where stem cells are stored. Carbon-based films were deposited on reference substrates using a magnetron sputtering technique. Amorphous carbon and diamond like carbon with anode laser source were studied in the work. Niche-like structures were designed to catch stem cells from blood flow and prevent their rapid differentiation. The blood-material interaction under dynamic conditions was performed and 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. The potential for endothelialisation was adjusted using human umbilical vein endothelium cells. The contact angle measurement was performed giving initial output information for tests on blood. The biomimetic approach considered niche-like structures reconstruction using materials science. In the work we have developed coatings that could mobilize and influence the differentiation of vascular endothelial cells.

aInstitute of Metallurgy and Materials Science, Polish Academy of Sciences, 25 Reymonta Street, 30-059 Cracow, Poland. *corresponding author: [email protected] bJoanneum Research Forschungsges mbH, Institute of Surface Technologies and Photonics, Functional Surfaces, Leobner Strasse 94, A-8712 Niklasdorf, Austria cDepartment of Medicine, Jagiellonian University Medical College, Cracow, Poland dDepartment of Biomedical Engineering, Koszalin University of Technology, Koszalin, Poland

I. INTRODUCTION forms a micro-environment that maintains cells and gives signals to the main cell for proliferation, maturation, and self-renewal. Cellular niches refer to an anatomical and functional structure, including cellular and extracellular components and local and systemic factors, that are integrated to regulate stem cell proliferation, differentiation, survival and localization [14-16]. In 1978, Schofield proposed the concept of the “stem cell niche” in studies of haematopoietic stem cells (HSCs) [17]. Since then, this hypothesis has been validated by a number of studies. The in vivo evidence of the existence of the stem cell niche was first provided in studies using invertebrate models [18] and in Drosophila germline stem cells [19, 20]. In mammals, stem cell niches have been identified in different tissues over the past several years, including bone marrow, brain tissues, hair follicles, the intestines, and teeth [21-26]. Theoretically, a stem cell niche is composed of the stem cells themselves along with stromal support cells, extra cellular matrix proteins, blood vessels and neural inputs. The interaction between biomaterial and stem cells is described in the literature [27-33].

The future and development of science can be seen in interdisciplinary areas of the field such as biomedical engineering. A great deal of research activity in the field of biomaterials development is focused on blood-material interaction. The developed materials will find their application in a new generation of cardiovascular devices especially cardiac support system. The best solutions are associated with the biomimetic surface of materials introduced into the human body. The combination of tissue engineering and materials science in the design of novel biomedical materials is now a worldwide trend [1-10]. In the treatment of cardiovascular diseases, tissue analogues reduce the risk of rejection and increase the chances for better and longer implantation with reduced immunosuppression support. A surface modification of polymers using thin layers allows for the formation of new features of materials while maintaining or slightly changing the physical properties of the applied polymer [11-13]. Each stem cell, as it grows and matures, has its own niche within a spatial structure of cells. It

REGENERATIVE NANOMEDICINE 2

Cell niches can be simulated in different ways. One model introduces controlled participation of stressors into the coating structure, which has been applied to the substrate of polymer implants used for cardiovascular regeneration. The surface modification of polymers using thin layers allows for the formation of new features of materials while maintaining or slightly changing the physical properties of the polymer [34]. The main focus of surface modification of medically used polymers is to improve the stability of the material that is in direct contact with tissue. Adhesion of layers to the substrate is one of the most important factors in classifying materials for biomedical engineering. To reconstruct the niche-like structures, the surface wrinkling was noted, appearing to reduce the overall strain energy [35-37]. The suggested explanation accounts for the common deformation of the substrate surface and the layer [38]. Description of the corrugation may be based on the determination of the creation of sinusoidal ridges on the surface that do not cause any loss of adhesion to the substrate. The main mechanism of wrinkling is joined with the substrate or subsurface areas with the applied layer uplifting. The reason for the folding of the deposited layer comes from the mechanical instability. This can be compared to the problem of instability of the elastic layer on a flexible substrate medium. In this model, both the base layers and the substrate have a biaxial stress. The result of compressive stress relaxation is the formation of the wavy structure on the surface [38-40]. Theoretical considerations of the folding effect of the soft layer-substrate showed that the folding mainly depends on the mechanical properties of the substrate and the interfacial area [38]. Our work was focused on cellular niche-like structures elaborated on surfaces of blood-contacting materials to influence the progenitor cells captured from the blood flow towards differentiation into endothelial cells. The purpose of the present study was to develop coatings that could mobilize and influence the differentiation of vascular endothelial cells. Simultaneously, the developed materials had to exhibit excellent haemocompatible properties, eliciting low thrombogenic and immune responses. Materials in the present study were selected with respect to the possibility of creating a suitable environment for the differentiation of endothelial cells under conditions of dynamic blood flow. The paper presents results of the process optimization regarding cellular niche-like structure formation based on the analysis of the microstructure and haemocompatibility performed in aortic flow conditions. Finally, the materials were verified using fluorescence analysis of endothelial growth and proliferation. The endothelium consists of cells that should be formed ultimately on the surface of newly designed materials from progenitor cells. The main factors of cellular differentiation are related to the mechanical and structural properties of the materials. Novelty of the presented work was to reconstruct the structure of the cell niches.

II. MATERIALS AND METHODS

1. Coating preparation

Coating deposition occurred by high-vacuum technologies on industrial-scaled coating equipment (chamber size 700x700x700 mm³) without heating of the substrate which remained at room temperature (25-30°C) to prevent any thermal damage of the polyurethane (PU). Before deposition, PU substrates were washed in isopropanol ultrasonically to remove all organic contaminations and subsequently dried for >48 h under fine vacuum conditions (<10 Pa) to remove water or alcohol content from manufacturing and/or cleaning.Afterwards, the samples were introduced under lamellar flow conditions to prevent any particle deposition on surfaces in the high-vacuum coating equipment and mounted 120 mm away from the deposition sources. The appropriate vacuum 2x10-

5 was reached by pumping with turbomolecular pump. They were the start conditions for the ion pretreatment of the surfaces using anode layer source plasma (ALS, ALS 340, Veeco Inc. Fort Collins (CO)). Pretreatment was performed in order to ensure better adhesion of the coating.Therefore, Ar and O2 were used in mixtures, while the substrates were continuously rotated on the planetary. This 1-dimensional rotation was not stopped until the end of the following deposition.For deposition, we used 2 different processes. (1) Magnetron sputtering was performed from highly unbalanced rectangular sputter targets (electrographite, 99.8% purity) in Ar atmosphere (5x10-3 mbar). N doping of the diamond-like carbon (DLC, a-C:H) coatings was reached by mixing nitrogen (N2) to the recipient atmosphere. For higher energetic deposition conditions, sputtering at ½ pressure was performed. (2) Plasma enhanced chemical vapour deposition (PECVD) was performed at 2x10-4 mbar pressure, but in acetylene atmosphere (C2H2) using the ALS.

Table 1 The composition and thickness of coatings deposited on PU substrate.

Coating material / process ThicknessHydrogenated amorphous carbon (a-C:H) (sputtered)

110nm

Hydrogenated amorphous carbon (a-C:H) (sputtered)

60nm

Hydrogenated amorphous carbon a-C:H (lower gas flow; sputtered) 100nm

Nitrogen-doped amorphous carbon a-C:N (sputtered)

105nm

Diamond like carbon (DLC) (deposited with Anode Laser Source ALS)

85nm


43nm


65nm

Diamond like carbon (DLC) (deposited with Anode Laser Source ALS) lower gas flow

86nm


After reaching the desired film thickness (see Table 1), the process was stopped, and the recipient was filled with nitrogen to reach atmospheric pressure and to remove the samples under laminar flow.

2. Microstructure characterisation

The microstructure characterization of the deposited coatings was performed using a TECNAI G2 FEG (200 kV) transmission electron microscope (TEM) equipped with a field emission gun (FEG). Thin foils for TEM observation were prepared for one sample in each group of materials using the focused ion beam (FIB) technique. For FIB preparation, a Quanta 200 3D Dual-Beam was used. A detailed description of the TEM microstructure analysis has been previously reported [41]. Phase analysis was performed using electron diffraction patterns and was confirmed through identification of high-resolution images (HR-TEM).

3. Blood-material interaction

The analysis of blood-material interactions was performed using a commercially available tester, Impact-R (Diamed) [42]. Expression of platelet activation markers was measured on CD61 gated objects using PAC-1 antibody for conformational change of glycoprotein IIb/IIIa and using CD62P (purchased in Abcam [AK4], prediluted (FITC) (ab134397)) for P-selectin. Integrated fluorescence of the activation marker was calculated as a multiplication total of geometric mean fluorescence by percentage of marker-positive objects. Aggregates of platelets were analysed after erythrocyte lysis by mixing 25 μL of blood with 0.4 mL FLS and subsequent fixation by addition of 3.5 mL to 1% paraformaldehyde in PBS. The cellular material was recovered by centrifugation (1000 g, 7 minutes) and immunostained (25 μL aliquots) with 4 μL Peridinin chlorophyll PerCP-CD14 (purchased in BD Biosciences Catalog No. 340585) and 5 μL FITC-CD61 (purchased in BD Biosciences Catalog No.555753) or 5 μL Fluorescein isothiocyanate (FITC)- CD61 alone for 30 minutes at room temperature. Samples were then washed in PBS and analysed by the flow cytometer. The percentage of granulocyte-platelet aggregates (leukocytes stained with platelet marker Integrin beta-3 cluster of differentiation61 (CD61)) was calculated using granulocyte forward/side scatter gate and additional monocyte cluster of differentiation 14 (CD14+) gate. The absolute number of platelets was calculated as the number of CD61 positive objects in reference to total granulocyte count. Small and large platelet aggregates were counted using forward/side scatter gates for CD61 positive objects in the flow cytometer analysis. Samples were washed in PBS solution three times and analysed using confocal laser scanning microscopy Carl Zeiss Exciter 5. Specific monoclonal antibodies were used to analyse the adhesion of active platelets by using two antibodies: P-selectin conjugated with fluorescein isothiocyanate (FITC) and (platelets with the active receptor selectin

P) human monoclonal antibodies protein tyrosine phosphatase, claster of differentiation 45(CD45) PE (purchased in BD Pharmingen™ Catalog No. 557748) conjugated with Phycoerythrin (PE)-Texas Red (active leukocytes). Thrombogenic potential of blood plasma was measured using a Zymuphen MP-activity ELISA kit (Hyphen Biomed, Eragny, France Code: A521096), according to the manufacturer’s instruction. This assay is based on the trapping of phospholipid-rich microparticles derived from cell membranes, using immobilized annexin V, followed by reconstitution of thrombin activity with calibrated clotting factors solution. Proteolytic activity of generated thrombin against the chromogenic substrate strictly correlates with the concentration of microparticles present in the blood plasma. Microparticles (MP) of the cell membranes are also known as microbubbles and range in size from 0.2 to 2.0 μm. They are released from the plasma membranes of all the cellular components of blood. Microparticles have no nucleus. On their surface are surface antigens that are characteristic of the cells from which they originate. The release of MP from eukaryotic cells is a normal physiological process that occurs during maturation and cell ageing [43, 44]. Increased release of MP can take place upon the activation of cells, for example, by binding blood components or immune complexes, cytokines, chemokines, stress factors, such as temperature, changes in osmotic pressure, tension generated during flow in vessels or under the influence of factors leading to apoptosis.Statistical analysis was performed using SPSS version 17.0 statistical package (SPSS Inc., Chicago, IL, USA) for personal computers. SPSS Statistics is a software package used for statistical analysis. Companion products in the same family are used for survey authoring and deployment (IBM SPSS Data Collection), data mining (IBM SPSS Modeler), text analytics, and collaboration and deployment (batch and automated scoring services). Data are presented as means ± SD (standard deviation) and ranges. Statistical significance was assumed for type-I error p < 0.05.

4. Endothelialisation

Human umbilical endothelial cells (HUVECs) were cultured for the experiment to assess the surface impact on endothelialisation. Human umbilical vein endothelial cells were purchased from Lonza (Catalog No. CC-2517 Swiss multinational, chemicals and biotechnology company, head-quartered in Basel). Each vial had a concentration of 500000 cells/mL. The cells were stored in liquid nitrogen until use. Approximately, 100000–125000 cells were plated in a 25 cm2 flask. From each vial, it was possible to prepare 4 or 5 flasks. Cells were resuspended in an endothelial cell culture basal medium mixed with cell growth and survival supplements (bullet kit growth mixture purchased from Lonza, including cell growth promoting serum, vitamins, and antibiotics,


optimized for HUVECs cat. no. CC-2517 and CC-2519). Before adding cells, the medium was warmed in a 37°C water bath. Cells were taken from the liquid nitrogen container and placed into a 37 °C water bath for 2–3 min. Under the laminar airflow chamber, a maximum of 1 mL of medium mixed with the added bullet kit. Everything was then pipetted into a 15 mL Falcon tube and diluted to 4 or 5 mL to receive 100000 or 125000 cells/flask, respectively. The resuspended cells were taken in the amount of 1 mm from the Falcon tube and introduced into a 25 cm2 cultivation flask. Then, each flask was filled up to 6 mL of medium with supplements. Cells were incubated in 37 °C, 5% CO2 and 100% humidity.

III. RESULTS

The results show the work that was performed in the context of haemocompatibility and the separate assessment of the potential for endothelialisation.

1. Microstructure and surface topography characterization

Microstructure analysis was performed in bright field mode. Transition electron microscopy (TEM) observations were performed on a cross-section of the amorphous carbon (a-C:H) 100 nm coating deposited on the polymer.The folds have a size characterized in the micrometre scale. No cracks in the surface were observed (Fig. 1).Selected area electron diffraction pattern demon-strated the presence of an amorphous structure of the coating. This is important because of the possibility of transferring large values of residual stress. Accumulation of stress in the amorphous structure can cause wrinkling without the danger of surface cracks appearing.

2. Blood-material interaction under dynamic conditions

Taking into consideration both activation of platelets and blood during dynamic testing and adhesion of blood cells to artificial surfaces, the analysis enables accurate assessment of blood-material interactions. Activation of platelets and their aggregation in blood collected after dynamic testing and the adhesion of active platelets (PLT) and leukocytes to surfaces were analysed separately. Analysis of the quality of blood through the techniques of flow cytometry is presented in Fig. 2 and Fig. 3. Fig. 2 presents the results of the platelet aggregate (PLT-AGR) formation on the materials subjected to analysis. The study focused on two types of aggregates, small (SMALL PLT-AGG) and large (BIG PLT AGR). Small platelet aggregates were defined as two plates, while large aggregates were defined as more than two plates. Fig. 3 refers to the consumption of the platelets.

Figure 2 Platelet aggregate versus material. PLT-AGR- overall platelet aggregates; SMALL PLT-AGG- small platelet aggregates; BIG PLT AGR- big platelet aggregates; DLC = a-C:H Plasma-enhanced chemical vapour deposition = (DLCALS) (according to the Table 1).

Figure 1 TEM Microstructure characterization of the a-C:H coating and the analysis of the degree of folding induced by the plasma-enhanced chemical vapour deposition.


Figure 3 Platelets consumption by cell lysis under shear forces; PLT %- % of consumed platelets; DLC = a-C:H Plasma-enhanced chemical vapour deposition = (DLCALS) (according to the Table 1).

A result of 100% PLT indicates no platelets were used and such a result was recorded as a negative control (bas). The positive control was obtained by blood association with adenosine di-phosphate (ADP). ADP is the strongest PLT activator. In this case, the strongest consumption occurred. The parameters of the PLT consumption of the analysed materials should be found between the parameters of the negative and positive control; the closer to the negative control the better. In comparison with the control material, all surfaces have a weaker activation of platelets except for the a-C:H 100 nm and DLC 65 nm samples. These two samples favour the formation of platelet aggregates both small (SMALL PLT-AGG%; consisted of two cells) and large (BIG PLT-AGR%; consist of more than two cells). For other coatings, small differences in platelet activation could be seen, but in all cases there is no observation of large aggregate (BIG PLT-AGG) formation. Adhesion of active platelets to material surfaces was smaller for PU modified with different coatings than the control sample (Fig. 4).

Figure 4 The relative amount of activated blood cell counts (platelets, leukocytes) in the function of the coating; N/N0- the relative value of the number of cells; DLC = a-C:H Plasma-enhanced chemical vapour deposition = (DLCALS) (according to the Table 1).

3. Microparticles

Microparticles (MPs) are small membrane vesicles

that are released by activated or apoptotic cells. The results showed that the release of MP (CD41+) from whole blood was higher for DLC-type coatings than whole MP stored in direct contact with the a-C:H. Increased release of MP is not only a result of contact with the artificial surface but also a result of blood donor. Generally, the material dedicated to contact with blood should not affect the activation processes of blood. Fig. 5 shows the dependence of the platelet consumption versus the amount of platelet microparticles.On basis of the studies, a classification of the materials according to their influence on the blood activation process was done. The material influence on the activation process and the creation of platelets derived microparticles were compared with the positive and negative (bas). The positive control is obtained by combining whole blood with adenosine di-phosphate. The negative control is obtained by associating anti-coagulated blood with sodium citrate or heparin. A large number of generated microparticles and high platelet consumption eliminates a potential biomaterial from further consideration. The best properties of the demonstrated layers based on a-C:H generating a low amount of microparticles with low platelet consumption is shown in Fig. 5.

Figure 5 Platelets consumption in the function of microparticles concentration in blood after dynamic test; Bas- negative control, ADP- adenosine di phosphate- positive control; DLC = a-C:H Plasma-enhanced chemical vapour deposition = (DLCALS) (according to the Table 1).

4. Endothelial cell proliferation

There were differences in the roughness of the surface types that is also strongly reflected in the colonization of the surface and its potential to rise. Coatings based on amorphous carbon better enhance the efficiency of endothelial monolayer formation. The most efficient cell growth was observed on the surfaces type a-C:H (sputtered) with a thickness of 105-110 nm. The following analysis (Fig. 6) shows the endothelial cell monolayer still in the forming stage, which can be recognized by the characteristic elongated pattern of the cells. The final stage of the endothelium layer exhibits the cube-like shape of the cells. None of the materials influenced effectively enough to form a full confluence.


IV. DISCUSSION

Surface functionalisation for blood-contacting purposes is a challenging problem for regenerative medicine. The idea is promising, but there are many difficulties involved in biomimetic surface design. The structures found in nature seem to be most helpful in decreasing the pain of the patient suffering from heart or circulatory system dysfunction. The coating systems presented in the initial research work attempted to create surface functionalisation for endothelial cell cultivation. The first surface modification was focused on haemocompatibility, antibacterial properties and the ability to generate an appropriate surface charge. All three of these functions were achieved by one type of material, which was applied as a thin coating directly

onto the polyurethane substrate. Conventionally used elements with antibacterial properties would not prevent clot formation. Furthermore, surface charge played a crucial role in surface porosity, which was the next step in novel biomaterial surface design. As described, the surface porosity provided the proper environment for endothelial cell seeding. We were able to design, propose, and prepare the appropriate pre-treatment of the surface using a coating that was only a few nanometres thick. The best parameters were found when using a Ti(C,N) coating. The phase composition is not enough to achieve a successful coating. It was necessary to adjust the thickness and the model of thin coating gas-phase nucleation. In general, it was observed that coatings in the thickness range of 5-30 nm demonstrated elastic properties.

Figure 6 Formation of endothelial layer on surfaces. Cell cytoskeleton was stained using Alexa Phalloidin 488 and nuclei by DAPI; DLC = a-C:H Plasma-enhanced chemical vapour deposition = (DLCALS) (according to the Table 1).


The adjusted coatings were finally deposited with the so-called initial model of thin film nucleation. The idea for the next biomaterial design step was found in nature, as well. The luminal side of a blood vessel is covered with endothelial cells. This layer is in direct contact with the bloodstream and under normal physiological conditions was able to effectively inhibit the activation of the coagulation system. The results of the study designed to evaluate the performance of an alternative in vitro dynamic test for haemocompatibility showed that surface functionalisation with a porous extracellular-like structure decreased the number of activated platelets and aggregates in comparison to non-porous structures and the endothelial cells on the surface. The idea to restore the structure and function of cell niche using the methods of materials engineering is of the great interest among bioengineers recently [45, 46]. Following the information given elsewhere [47] varying microenvironments affecting the modulation of cell behaviours may influence on specific cell selection [48-50]. In order to reproduce the structure of the cell niche the authors Chi et al [51] proposed polyelectrolyte multilayers (PEM) which allows the cells to self-assemble on a surface resulting in the growth of a film and variation of surface properties. PEM films in the cited article have been used to study boost progenitor cells behaviour [52-54]. Preliminary studies for this work were carried out under the attempt to create microenvironment for cells in the form of wells, made by laser ablation [55] and the in the form of wrinkles [56]. The method of the residual stress analysis was elaborated [57]. The proposed wrinkle structure was developed by Lackner et al. [58]. The authors underlined that the high intrinsic film stresses due to high energetic film growth and the huge difference in elastic properties of films and polymer substrates result in hierarchical and self-adapting nanowrinkling phenomena. Intrinsic stresses are a reason for the hierarchical and self-adapting wrinkling of human skin too, being mechanically treated quite similar to the coated polymers as a hard layer on a soft foundation. Mechanically, the wrinkles have high impact on the elongation behaviour: In the elastic region of the stress–strain curve, they are smoothing out at increasing strain [59- 66].The proposed surface modification was intended to create a substrate with haemocompatible features. The influence of the anode layer source caused the wrinkles on the surface. The microstructure analysis revealed that the main mechanism of the fold creation was the high energetic process based on the plasma-enhanced chemical vapour deposition. Following the requirements defined by the Regional Blood Donor Centre, haemolysis in stored blood should not exceed 0.8% of the weight of the red blood cells. Increased haemolysis in the dynamic system was not only the result induced by contact between the blood and the biomaterial but also by other factors such as high rotation per minute in the

dynamic test on blood. It should also be noted that circulation was conducted in a closed system and that the volume of the circulating blood was small (approximately 1 mL). In all cases the surface modification improved the haemocompatible properties. Microparticle activity of blood plasma denotes the fragmentation of platelets and the release of cell membrane vesicles. Microparticles impose thrombotic burden on circulating blood [67] and were recently found to correlate with atherosclerosis and a risk for major adverse cardiovascular events, such as heart infarction or stroke [68, 69]. In analysing the results, it is also important to note that the values of the free haemoglobin levels were within the range of the specified standards developed for the preparation and storage of whole blood. A decrease in the number of platelets in the platelet consumption analysis can be caused by the activation and formation of platelet-platelet and platelet-leukocyte aggregates adhering to the surface. The present study investigated new materials for coating cardiovascular implant surfaces for application in forced blood-circulation systems, with consideration given to the design, processing and testing of relevant parameters. New materials for these devices that exhibit improved blood-material interactions are urgently needed. Therefore, the main objective of this work was to effectively create an endothelium on the surface. This concept is borrowed from the layered structure of the blood vessel, where the endothelial surface is in direct contact with blood, effectively inhibiting coagulation processes. The efficiency of the endothelium monolayer formation was analysed using vascular endothelial cells, which are commercially available. The good properties of the materials based on amorphous carbon were confirmed for both contact with blood and the appropriate substrate for endothelium formation. The coatings of this type induced the slightest aggregation and activation processes of blood. The final selection was made by comparing the amount of apoptotic origin microparticles formed as a function of platelet consumption. Materials should generate the least microparticles with a minimum consumption of platelets [70]. Coatings based on amorphous carbon with a thickness of 100-110 nm were found to exhibit low values of both of these parameters. As a reference, negative and positive controls were prepared and, based on these controls, the final selection of materials was performed. Further work, which will be a scientific series of the present publication will cover the uptake of stem cells from whole blood and their differentiation towards endothelial cells, mainly through surface topography and microstructure of the substrate.

V. CONCLUSIONS

- The anode laser source has enabled the


elaboration nano- and microwrinkles on the surface in the controlled way. The wrinkles could not generate cracks that lead to delamination of the coating from the substrate. The coatings have been optimized by means of transmission electron microscopy. It has been found that the structuring of the surface improved the monolayer efficiency formation of vascular endothelium. Such effect was desirable to inhibit the coagulation process.

- All tested materials were considered within the limits of acceptability between positive and negative control. The best properties were obtained for the coatings which were deposited using the anode laser source. Tests were conducted in hydrodynamic conditions using whole human blood.

- The process of the surface structuring in the form of nano- and microwrinkles contributed to reduction of platelet consumption under dynamic conditions. The results showed decreased activation of the platelet receptor in the attached to the surface platelets

- The final selection of the materials was based on the analysis of the consumption of the blood plates versus amount of platelet-derived microparticles. This type of analysis is reliable and allows to predict the activation process and the destruction of the cellular components in the direct contact with artificial materials. Based on the tests of the microparticles we have selected materials which could be considered clinically.

VI. ACKNOWLEDGEMENTS

The research was financially supported by the Project no. 2014/13/B/ST8/04287 “Bio-inspired thin film materials with the controlled contribution of the residual stress in terms of the restoration of stem cells microenvironment” of the Polish National Centre of Science.

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Biomimmetic surfaces effectively inhibiting coagulation ... · journal eage: ww.necentralress ... The work focused on developing coatings with niche-like surfaces that resemble stem