Clinical Hemorheology and Microcirculation - Volume 52, issue 2-4

Authors: Krüger, A. | Braune, S. | Kratz, K. | Lendlein, A. | Jung, F.

Article Type: Research Article

Abstract: Background: The patency of small-diameter vascular prostheses is limited by several factors such as thrombogenicity, which is strongly influenced by surface roughness and chemical composition, or a mechanical mismatch between the elastic modulus of an artery and of the vascular prosthesis. A confluent layer of endothelial cells onto the inner surface of vascular prostheses could improve the hemocompatibility of the device. Biomaterials with adjustable elastic properties could be tailored to the values of human arteries so that a prothesis mismatch could be avoided. It was recently demonstrated that a co-culture of endothelial cells with angiogenically stimulated monocytes (aMO2) shows an …accelerated formation of a functional confluent endothelial cell monolayer on soft hydrophobic poly(n-butyl acrylate) (cPnBA) networks. In addition, the cell compatibility with vascular smooth muscle cells and aortic fibroblasts, which are other important cell types of the vessel wall, is essential for a vascular prosthesis material and must therefore be explored. Purpose: Here we investigated the interaction of human vascular smooth muscle cells and aortic fibroblasts with cPnBA04 and cPnBA73. Material and methods: Human primary vascular smooth muscle cells and aortic fibroblasts were seeded on the two cPnBAs with different elastic moduli (cPnBA04 - 250 kPa and cPnBA73 - 1100 kPa) over 72 h. A live-dead staining (fluorescein diacetate/propium iodide) was performed to determine the morphology and viability of adherent cells. Furthermore, the extracellular matrix components, the actin cytoskeleton, the cell-material-contacts and the cytokine profiles were analysed. Results: Both cell types adhered and were viable on cPnBA04 and cPnBA73. The level of pro-inflammatory cytokine secretion (IFN-γ and TNF-α) by smooth muscle cells and vascular fibroblasts was comparable to that of cells cultivated on a control material. The release of these cytokines by human fibroblasts was higher on cPnBA73 compared to cPnBA04. Both cell types secreted an extracellular matrix comparable to cells seeded on a control material. Conclusion: The study revealed, that cPnBA with varying elastic moduli are not only suitable for the cultivation of endothelial cells, but also for human vascular smooth muscle cells and aortic fibroblasts. Therefore, cPnBA could be a potential candidate material for the development of cardiovascular prostheses. Show more

Keywords: Cardiovascular disease, atherosclerosis, polymer-based biomaterial, cPnBA

DOI: 10.3233/CH-2012-1605

Citation: Clinical Hemorheology and Microcirculation, vol. 52, no. 2-4, pp. 283-294, 2012

Authors: Scharnagl, N. | Hiebl, B. | Trescher, K. | Zierke, M. | Behl, M. | Kratz, K. | Jung, F. | Lendlein, A.

Article Type: Research Article

Abstract: The chemical composition of a substrate can influence the adhesion, viability and proliferation of cells seeded on the substrate. The aim of this work was to investigate the influence of different cationic or anionic moieties in acrylonitrile-based copolymers on the interaction with fibroblasts. A series of ten different types of acrylonitrile-based copolymers with a random sequence structure was prepared using a water born synthesis process to exclude potential residues of organic solvents. As charged comonomers cationic methacrylic acid-2-aminoethylester hydrochloride (AEMA), N-3-amino-propyl-methacrylamide hydrochloride (APMA) and anionic 2-methyl-2-propene-1-sulfonic acid sodium salt (NaMAS) were utilized. By application of a specific sintering procedure the …copolymer materials were processed into transparent disks for conducting cell tests in direct contact. The copolymers were analyzed with respect to their composition and surface properties. Cytotoxicity tests of the polymer extracts, as well as of the disks were performed with L929 mouse fibroblasts. All copolymers showed no cytotoxic effects. Furthermore, for higher molar ratios of AEMA an increase in cell growth could be observed, which might be a hint that higher charge densities are favorable for the proliferation of L929 cells. Show more

Keywords: Acrylonitrile, copolymers, fibroblasts, surface charges, biocompatibility

DOI: 10.3233/CH-2012-1606

Citation: Clinical Hemorheology and Microcirculation, vol. 52, no. 2-4, pp. 295-311, 2012

Authors: Rüder, Constantin | Sauter, Tilman | Kratz, Karl | Peter, Jan | Jung, Friedrich | Lendlein, Andreas | Zohlnhöfer, Dietlind

Article Type: Research Article

Abstract: Stent thrombosis and restenosis after drug-eluting stent (DES) implantation remains a relevant problem in the cardiovascular field. The polymer-based biomaterial (e.g. stent coating) requirements are comprehensive, since the polymeric material ideally should ensure an effective re-endothelialization by recruiting endothelial cells (EC) and endothelial progenitor cells (EPC). Simultaneously, the polymer should effectively prevent adherence of smooth muscle cells (SMC) and thereby inhibiting restenosis. The aim of this study was to gain a basic understanding on the interaction of SMC and human umbilical vein endothelial cells (HUVEC) with nonporous polymer films. A multifunctional copolyetheresterurethane (PDC) was chosen as candidate material: PDC consists …of poly(p-dioxanone) (PPDO) and poly(ε-caprolactone)-segments (PCL). In our study it was compared to the degradable PPDO homopolymer and poly(vinylidene fluoride-co-hexafluoropropene) (PVDF), an established coating material of DES in clinical applications intended for longterm applications. The films were analyzed according to their thermomechanical and surface properties before being examined in contact with HUVEC and SMC concerning cell viability, proliferation and adhesion. Experimental results showed that adhesion could be improved for HUVEC on PDC compared to PPDO and PVDF. In contrast, SMC attachment is largely suppressed on PDC polymeric films indicating a cell-specific response of HUVEC towards PDC. In conclusion, PDC represents a promising candidate material for future cardiovascular applications like e.g. biodegradable (PDC) stent coatings. Show more

Keywords: Endothelialization, drug eluting stent, degradable polymer, cell selectivity

DOI: 10.3233/CH-2012-1607

Citation: Clinical Hemorheology and Microcirculation, vol. 52, no. 2-4, pp. 313-323, 2012

Authors: Schneider, Tobias | Kohl, Benjamin | Sauter, Tilman | Kratz, Karl | Lendlein, Andreas | Ertel, Wolfgang | Schulze-Tanzil, Gundula

Article Type: Research Article

Abstract: Degradable polymers with a tailorable degradation rate might be promising candidate materials for biomaterial-based cartilage repair. In view of the poor intrinsic healing capability of cartilage, implantation of autologous chondrocytes seeded on a biocompatible slow degrading polymer might be an encouraging approach to improve cartilage repair in the future. This study was undertaken to test if the fiber orientation (random versus aligned) of two different degradable polymers and a polymer intended for long term applications could influence primary articular chondrocytes growth and ultrastructure. A degradable copoly(ether)esterurethane (PDC) was synthesized via co-condensation of poly(p-dioxanone)diol and poly(ε-caprolactone)diol using an aliphatic diisocyanate as …linker. Poly(p-dioxanone) (PPDO) was applied as commercially available degradable polymer, while polyetherimide (PEI) was chosen as biomaterial enabling surface functionalization. The fibrous scaffolds of PDC and PPDO were obtained by electrospinning using 1,1,1,3,3,3 hexafluoro-2-propanol (HFP), while for PEI dimethyl acetamide (DMAc) was applied as solvent. Primary porcine articular chondrocytes were seeded at different cell densities on the fibrous polymer scaffolds and analyzed for viability (fluorescein diacetate/ethidiumbromide staining), for type II collagen synthesis (immunolabelling), ultrastructure and orientation on the fibers (SEM: scanning electron microscopy). Vital chondrocytes adhered on all electrospun scaffolds irrespective of random and aligned topologies. In addition, the chondrocytes produced the cartilage-specific type II collagen on all tested polymer topologies suggesting their differentiated functions. SEM revealed an almost flattened chondrocytes shape on scaffolds with random fiber orientation: whereby chondrocytes growth remained mainly restricted to the scaffold surface. On aligned fibers the chondrocytes exhibited a more spindle-shaped morphology with rougher cell surfaces but only a minority of the cells aligned according to the fibers. As a next step the reduction of the fiber diameter of electrospun scaffolds should be addressed as an important parameter to mimic cartilage ECM structure. Show more

Keywords: Chondrocytes, electrospinning, scaffold, differentiation, multiblock copolymer

DOI: 10.3233/CH-2012-1608

Citation: Clinical Hemorheology and Microcirculation, vol. 52, no. 2-4, pp. 325-336, 2012

Authors: Tzoneva, Rumiana | Seifert, Barbara | Behl, Marc | Lendlein, Andreas

Article Type: Research Article

Abstract: Hemocompatibility of elastic multiblock copolymers PDC, based on poly(p-dioxanone) (PPDO)/poly(ε-caprolactone)-segments, capable of a shape-memory effect, and PDD, based on PPDO/poly((adipinate-alt-1,4-butanediol)-co-(adipinate-alt-ethylene glycol)-co-adipinate-alt-diethylene glycol)-segments, was studied in order to assess their suitability for an application aiming at blood vessels regeneration. The results were compared with polypropylene (PP) which is a widely used blood-contacting material for devices as blood oxygenators and dialysis tubes. Protein adsorption studies showed diverse blood plasma proteins in a relatively high amount on both elastic polymers compared to the poor amount of plasma proteins adsorbed on PP. Study of the coagulation system revealed high thrombin formation on PDC and …no difference in plasma kallikrein activation between elastic multiblock copolymers and the reference PP. Activation of complement system was higher for PDC followed by PDD and lower for PP. However, platelet adhesion and activation were hardly suppressed on the multiblock copolymers compared to the PP surface, where the number of adhered platelets and the activation rate were significant. The present results reveal that the tested multiblock copolymers with improved elastic properties and shape-memory capability (PDC) show low thrombogenicity and are promising candidates for vascular tissue engineering. Show more

Keywords: Hemocompatibility, degradable elastic polymer, protein adsorption, coagulation, thrombin formation, platelet adhesion/activation, shape-memory polymer

DOI: 10.3233/CH-2012-1609

Citation: Clinical Hemorheology and Microcirculation, vol. 52, no. 2-4, pp. 337-348, 2012

Authors: Braune, S. | Alagöz, G. | Seifert, B. | Lendlein, A. | Jung, F.

Article Type: Research Article

Abstract: A dataset of 439 confocal laser scanning microscopic images was analyzed to investigate the potential of an image-based automated analysis for identifying and assessing adherent thrombocytes on polymer surfaces. Parameters for image optimization of glutardialdehyde induced fluorescence images were classified and data mining was performed using the Java image processing software ImageJ. Previously reported analysis required that each thrombocyte had to be identified interactively and outlined manually. Now, we were able to determine the number and area of adherent thrombocytes with high accuracy (spearman correlation coefficient r = 0.98 and r = 0.99) using a two-stage filter-set, including a rolling …ball background subtraction- and a watershed segmentation-algorithm. Furthermore, we could proof a significant correlation between these parameters (spearman correlation coefficient r = 0.97), determining both as suitable predictors for the evaluation of material induced thrombogenicity. The here reported image-based automated analysis can be successfully applied to identify and measure adherent thrombocytes on polymer surfaces and, thus, might be successfully integrated in a high-throughput screening process to evaluate biomaterial hemocompatibility. Show more

Keywords: Hemocompatibility, thrombocytes, biomaterial, high-throughput, image-based automation

DOI: 10.3233/CH-2012-1610

Citation: Clinical Hemorheology and Microcirculation, vol. 52, no. 2-4, pp. 349-355, 2012

Authors: Wang, Weiwei | Ma, Nan | Kratz, Karl | Xu, Xun | Li, Zhengdong | Roch, Toralf | Bieback, Karen | Jung, Friedrich | Lendlein, Andreas

Article Type: Research Article

Abstract: Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into a variety of cell types. Therefore, they are widely explored in regenerative medicine. The interaction of MSCs with biomaterials is of great importance for cell proliferation, differentiation and function, and can be strongly influenced by numerous factors, such as the chemical nature and the mechanical properties of the material surface. In this study, we investigated the interaction of bone marrow derived human MSCs with different amorphous and transparent polymers namely polystyrene (PS), polycarbonate (PC), poly(ether imide) (PEI), polyetherurethane (PEU) and poly(styrene-co-acrylonitrile) (PSAN). To ensure that the MSCs were …solely in contact to the testing material we applied polymeric inserts, which were prepared from the aforementioned polymers via injection molding. The explored inserts exhibited a similar wettability with advancing contact angles ranging from 84 ± 7° (PEU) to 99 ± 5° (PS) and a surface roughness of Rq ≤ 0.86 μm. The micromechanical properties determined by AFM indentation varied from 6 ± 1 GPa (PEU) to 24 ± 5 GPa (PSAN). Cells presented different adhesion rates on the polymer surfaces 24 hours after seeding (45 ± 7% (PS), 63 ± 1% (PC), 75 ± 4% (PEI), 69 ± 2% (PEU) and 61 ± 5% (PSAN)). The cells could proliferate on the polymer surfaces, and the fold change of cell number after 16 days of culture reached to 1.93 ± 0.07 (PS), 3.38 ± 0.11 (PC), 3.65 ± 0.04 (PEI), 2.24 ± 0.15 (PEU) and 3.36 ± 0.09 (PSAN). Differences in cell apoptosis could be observed during the culture. After 7 days, the apoptosis of cells on PC, PEI and PSAN decreased to a level comparable to that on standard tissue culture plate (TCP). All of the tested polymers exhibited low cytotoxicity and allowed high cell viability. Compared to cells on TCP, cells on PC and PEI showed similar morphology, distribution as well as F-actin cytoskeleton organization, whereas cells on PSAN were distributed less evenly and cells on PEU were less oriented. Cells were more likely to form clusters on PS. Conclusively, we demonstrated the influence of polymer substrates on the cellular behaviour of MSCs, which could be included in the development of novel design concepts based on polymeric biomaterials. Show more

Keywords: Mesenchymal stem cells, polymer surface, cell-material interaction, cell adhesion, proliferation, apoptosis

DOI: 10.3233/CH-2012-1611

Citation: Clinical Hemorheology and Microcirculation, vol. 52, no. 2-4, pp. 357-373, 2012

Authors: Roch, Toralf | Krüger, Anne | Kratz, Karl | Ma, Nan | Jung, Friedrich | Lendlein, Andreas

Article Type: Research Article

Abstract: For the successful clinical and biological application of polymers, their interaction with cells, tissues, and body fluids has to be well characterized. In order to investigate how the physical, chemical, and mechanical properties of candidate biomaterials influence cell behaviours, the testing sample is usually placed in commercially available cell culture plates. Thus, not only the testing sample itself but also the culture dish material might influence the cell behaviour. Therefore, an insert system was created to exclude this influence and allow investigations of the testing material solely. In this study micropatterned inserts prepared from polystyrene (PS) as well as from …poly(ether imide) (PEI) with three different roughness levels of i) Rq = 0.12 μm (PS) and 0.23 μm (PEI); ii) Rq = 3.52 μm (PS) and 3.92 μm (PEI); and iii) Rq = 16.04 μm (PS) and 22.65 μm (PEI) were explored with regard of their immuno-compatibility including the determination of potential contaminations with endotoxins or other microbial products. The endotoxin levels of the inserts were determined to be less than 0.07 EU/mL, which is well below the U.S. Food and Drug Administration limit of 0.5 EU/mL and the survival of murine macrophages cultured in the inserts was not impaired. Activation of early immune mechanisms such as complement activation and the generation of reactive oxygen species could not be observed. All tested materials had no influence on the cytokine secretion from cells of whole human blood. The investigated inserts were immuno-compatible and apparently free of contaminations with microbial products. The roughness of the inserts had no stimulatory or inhibitory effect on early immune mechanisms. Conclusively, the 24-well plate insert systems introduced in this study allow investigating the interactions of tailored surface properties such as roughness with many other cell types, without the disadvantage of the standard commercially available culture vessels influencing the biomaterial testing. Show more

Keywords: Immuno-compatibility, endotoxins, surface roughness, biomaterial, polystyrene, poly(ether imide)

DOI: 10.3233/CH-2012-1612

Citation: Clinical Hemorheology and Microcirculation, vol. 52, no. 2-4, pp. 375-389, 2012

Authors: Trescher, Karoline | Scharnagl, Nico | Kratz, Karl | Roch, Toralf | Lendlein, Andreas | Jung, Friedrich

Article Type: Research Article

Abstract: As shown in several studies, various properties of biomaterials such as stiffness, surface roughness, chemical composition or the amount of functional groups at the surface can influence adhesion, viability, proliferation and functionalities of cells. The aim of this work was to explore whether a cell-selective effect could be achieved for acrylonitrile-based copolymers containing different contents of positively charged functional groups, which were introduced by incorporation of methacrylic acid-2-aminoethylester hydrochloride (AEMA) units. The p(AN-co-AEMA) copolymers were synthesized by suspension polymerization in water and processed into disk shaped test specimen via a sintering process to ensure the absence of organic solvents in …the copolymers. Copolymers with an AEMA content of 1.4, 1.6, and 4.4 mol-% were investigated according to their cell-selective capacity, which should support the adhesion, viability and proliferation of keratinocytes, while the adherence of fibroblasts should rather be disabled. The test samples were seeded with primary human keratinocytes and primary human dermal fibroblasts in mono- as well as in co-cultures. Tissue culture plate polystyrene (TCP) was used to control the physiologic growth of the cells. Density and viability of attached and non-adherent cells were analyzed by live/dead staining, lactate dehydrogenase (LDH) assay and flow cytometry with DAPI staining. For the assured discrimination of adherent cell types in coculture a keratin/vimentin-staining was performed. On copolymers with 4.4 mol-% AEMA adherent keratinocytes in monoculture and cocultured keratinocytes and fibroblasts showed a higher viability, a lower impairment of cell membranes and higher densities of viable cells compared to both other copolymers. For adherent fibroblasts these parameters did not differ between the copolymers and an increasing ratio of keratinocytes to fibroblasts in cocultures were found with increasing AEMA content. The results showed that keratinocytes and fibroblasts can be influenced by copolymers with different contents of positively charged functional groups. Since the tendency of a better adherence and viability of keratinocytes with increasing amounts of positively charged functional groups was shown, the potential enhancement by further increase of the amount of positively charged functional groups shall be tested in a future study. Show more

Keywords: Keratinocytes, fibroblasts, coculture, amine functional group, surface charge

DOI: 10.3233/CH-2012-1613

Citation: Clinical Hemorheology and Microcirculation, vol. 52, no. 2-4, pp. 391-401, 2012

Authors: Jung, F. | Schulz, C. | Blaschke, F. | Muller, D.N. | Mrowietz, C. | Franke, R.P. | Lendlein, A. | Schunck, W.-H.

Article Type: Research Article

Abstract: Epoxyeicosatrienoic acids (EETs) produced by cytochrome P450 (CYP)-dependent epoxidation of arachidonic acid (AA) inhibit thrombocyte adhesion to the vascular wall. Upon dietary omega-3 fatty acid supplementation, EETs are partially replaced by eicosapentaenoic acid (EPA)-derived epoxyeicosatetraenoic acids (EEQs) and docosahexaenoic acid (DHA)-derived epoxydocosapentaenoic acids (EDPs). We hypothesized that the omega-3 epoxy-metabolites may exhibit superior anti-thrombogenic properties compared to their AA-derived counterparts. To test this hypothesis, we analyzed the effects of 11,12-EET, 17,18-EEQ and 19,20-EDP on Ristocetin-induced thrombocyte aggregation (RITA), a process that mimics thrombocyte adhesion to the vascular wall. The eicosanoids were added for 5, 30, or 60 minutes to thrombocyte-rich …plasma freshly prepared immediately after blood collection from stringently selected apparently healthy subjects. Thrombocyte aggregation was then induced by Ristocetin (0.75 mg/mL) and assessed by turbidimetric measurements. After 60 minutes of preincubation, all three epoxy-metabolites significantly decreased the rate of RITA. 17,18-EEQ and 19,20-EDP were effective already at 1 μM, whereas 5-fold higher concentrations were required with 11,12-EET. Addition of AUDA, an inhibitor of the soluble epoxide hydrolase, potentiated the effect of 17,18-EEQ resulting in a significant further decrease of the velocity as well as amplitude of the aggregation process. In contrast to their profound effects on RITA, none of the epoxy-metabolites was effective in reducing collagen- or ADP-induced thrombocyte aggregation. These results indicate a highly specific role of CYP-eicosanoids in preventing thromboembolic events and suggest that the formation of 17,18-EEQ and 19,20-EDP may contribute to the anti-thrombotic effects of omega-3 fatty acids. Show more

DOI: 10.3233/CH-2012-1614

Citation: Clinical Hemorheology and Microcirculation, vol. 52, no. 2-4, pp. 403-416, 2012